Critical Review

Protein Self-modification by Heme-generated Reactive Species

Enrico Monzani, Stefania Nicolis, Raffaella Roncone, Marica Barbieri, Alessandro Granata

and Luigi CasellaDipartimento di Chimica Generale, Via Taramelli 12, Pavia, Italy

Summary

In the presence of H2O2, heme proteins form active inter-mediates, which are able to oxidize exogenous molecules. Oftenthese products are not stable compounds but reactive species ontheir own, such as organic radicals. They can both diffuse tothe bulk of the solution or react with the protein that generatedthem. Here, we describe the self-modification underwent byheme proteins with globin-type fold, that is, myoglobin, hemo-globin, and neuroglobin when treated with NO2

2or catechols

in the presence of H2O2. The reactive nitrogen species gener-ated by NO2

2 give rise to nitration, oxidation, and/or crosslink-ing reactions between the proteins or their subunits. The qui-nones formed upon reaction with catechols easily modify Cysand His residues and eventually cause protein aggregation,which induces precipitation. The pattern of modificationsundergone by the protein strongly depends on the nature of theprotein and the reaction conditions. � 2007 IUBMB

IUBMB Life, 60(1): 41–56, 2008

Keywords heme-generated reactive species; protein self-modifica-

tion; oxidation reactions; crosslinking reactions; myoglo-

bin; hemoglobin; neuroglobin.

Abbreviations Hb, hemoglobin; Mb, myoglobin; HMb, human myo-

globin; hhMb, horse heart Mb; Ngb, neuroglobin;

Cys-DA, cysteinyl-dopamine; DAQ, reactive quinone

and semiquinone species formed upon oxidation of

dopamine; fQ, fluoroquinone and/or the dimer resulting

from coupling of the quinone with 3-fluorophenol;

Gdn-HCl, guanidinium chloride; HPA, 3-(4-hydroxy-

phenyl)-propionic acid; RNS, reactive nitrogen species;

ROS, reactive oxygen species.

INTRODUCTION

The heme is a very versatile group present in the active site

of a large number of proteins exhibiting a variety of activities.

This prosthetic group is found in electron-transfer proteins,

such as cytochromes (1), in monooxygenases, such as cyto-

chrome P450 (2), in peroxidases (3), such as NO synthase (4),

in O2 storage and transport proteins, such as myoglobin (Mb)

and hemoglobin (Hb) (5), and in cytochrome c oxidase (6) in

which one heme acts as electron-transfer group while another,

together with a copper center, acts as O2 binding and reduction

site.

The activity of the heme is fine-regulated by the protein

backbone, which controls the coordination state of the iron

(five- or six-coordinated), the iron(III)/iron(II) redox potential,

the polarity around the prosthetic group and the accessibility of

the active site by exogenous molecules. In Mb and Hb, the pro-

tein scaffold is designed to have a close active site accessible to

O2; in peroxidases, the active site is much more accessible even

to large molecules (7, 8). P450s may display an active site very

selective for substrates or a relatively broad pocket, allowing

the binding of different substrates, according to the specific

function of the enzyme.

It is worth noting that despite this ‘‘fine regulation’’ exerted

by the protein residues close to the active site, heme proteins

very often show, besides their main activity, catalytic properties

in different processes. For example, the P450s are able to act

as peroxygenase enzymes in the presence of alkyl peroxides

through the so-called ‘‘peroxide shunt’’ (9). Even the electron-

transfer protein cytochrome c, which has a six-coordinated heme

and a very compact structure, when partially denaturated at

acidic pH is capable of oxidizing substrates with H2O2 (10, 11).

Interestingly, also for the well-known O2-binding heme-pro-

teins Mb and Hb, new activities have been reported through the

years for their deoxy, oxy, and met forms. One of the most im-

portant reactions is with NO because it may be responsible for

the inactivation of cytochrome c oxidase. The oxy form of Mb

and Hb reacts promptly (almost with a diffusion-limiting rate)

with NO, producing the met form and NO32, thus controlling

the NO concentration in the body (12–16). Also, the deoxy

form is able to react with NO, giving rise to nitrosyl adducts

(17, 18). At the same time, human myoglobin (HMb) and Hb

Address correspondence to: Luigi Casella, Dipartimento di Chimica

Generale, Via Taramelli 12, I-27100 Pavia, Italy. Tel:139-0382-987331.

Fax:139-0382-52854. E-mail: bioinorg@unipv.it

Received 5 October 2007; accepted 6 October 2007

ISSN 1521-6543 print/ISSN 1521-6551 online

DOI: 10.1002/iub.10

IUBMB Life, 60(1): 41–56, January 2008

are considered to be active in preserving NO bioactivity through

the formation of an S-nitrosothiol with the Cys110 residue of

HMb (19) or with the Cysb93 residue of Hb (20, 21).

In the presence of H2O2 (but also with other oxidants),

metHb and metMb generate highly reactive ferryl species,

FeIV5O, with a radical localized on the protein. These species

are capable of reacting with several substrates, for instance can

oxidize NO22 to NO2 (22–24), phenols (25) or catechols to rad-

ical species, sulfoxidize organic sulfides (26–30), and epoxidize

alkenes (27–30). Even though the catalytic activity of Mb and

Hb in these reactions is modest, their concentration in the body

makes these side activities of potential physiological relevance.

In addition, the protein active species may also self-react with

protein residues in an intramolecular process, or with another

protein in solution. For example, it is known that the treatment

of HMb with H2O2 induces protein dimerization (31, 32).

NO2 and ONOO2 are important reactive molecules gener-

ated by the heme-protein active species and capable of attacking

amino-acid residues. Both of them can nitrate proteins, a pro-

cess often observed in pathophysiological conditions (33). NO2

and ONOO2 can be formed by heme proteins through different

mechanisms, including the reaction of the heme-Fe-O2 form

with NO (15), that of the FeIV5O species with NO22 (23, 24,

34–36), and that of the met form with H2O2 in the presence of

NO22 (23, 24, 36–40). Another source of reactive products is

the reaction of catechols with H2O2 catalyzed by the met form

of heme proteins. The catalysis occurs through one-electron oxi-

dation steps, and the first product formed is a reactive semiqui-

none species. It may attack protein residues or dismutate into

the starting catechol and a quinone molecule. These quinones

are on their own reactive molecules susceptible to undergo

nucleophilic attack by polar groups such as those contained in

amino acids residues, thereby causing protein modification. Re-

active quinones (and semiquinones) will be generally labeled as

DAQ, making reference to those generated from dopamine, one

of the most important biological sources. Covalent adducts

between DAQ and proteins have been observed in several cases,

in particular with neuronal proteins such as a-synuclein, the

human dopamine transporter, tyrosine hydroxylase, and parkin

(41–51).

Herein, we describe the self-modification undergone by

heme proteins of globin type, that is, Mb (both HMb and from

horse heart, hhMb), Hb, and the recently discovered neuroglo-

bin (Ngb) when they react with NO22 or catechols in the pres-

ence of H2O2. The focus will be mostly on the results obtained

in our laboratory, but the relevant results obtained by other

groups will also be emphasized.

ACTIVATION OF NO22 BY HEME PROTEINS IN

THE PRESENCE OF H2O2: NITRATION OF Tyr ANDOXIDATION OF Cys RESIDUES

Reactive nitrogen species (RNS) derived from NO are

involved in many pathological conditions (52, 53). Although

NO performs many important physiological functions (4, 54),

its overproduction can have damaging effects (54, 55). A major

product of NO metabolism is NO22 (56), and markedly

increased NO22 levels have been detected in situations (e.g.,

during inflammatory processes), in which NO is overproduced

(57, 58). NO22 can be oxidized through different pathways,

causing the formation of RNS (59) that can nitrate free Tyr

(generating 3-O2N-Tyr) (60) and Tyr residues in proteins (61,

62). An important pathway is the reaction of NO22 with heme

peroxidases in the presence of H2O2 (peroxidase/NO22/H2O2

system), which has been the subject of several recent studies

(36, 60–64).

We and others proposed that the two competing mechanisms

exist for the peroxidase catalyzed phenol nitration in the pres-

ence of NO22/H2O2 (23, 24, 36, 39, 40, 65). The first one

involves one-electron oxidation of NO22 by the peroxide-gener-

ated enzyme intermediates known as compound I and

compound II (7, 66), generating NO2 (reactions 1–3, where E

represents the native FeIII form of the enzyme); NO2 could then

either nitrate a phenol with a reaction stoichiometry of 2:1

(reactions 4 and 5) or react directly with a peroxidase-generated

phenoxy radical (reaction 5):

Eþ H2O2 ! compound Iþ H2O (1)

compound Iþ NO2� ! compound IIþ NO2 (2)

compound IIþ NO2� þ 2Hþ ! Eþ NO2 þ H2O (3)

NO2 þ PhOH ! NO2� þ PhO� þ Hþ (4)

NO2 þ PhO� ! O2N-PhOH (5)

The second pathway involves initial binding of NO22 to the

heme-iron center of the protein (reaction 6), followed by reac-

tion of this complex with H2O2. The resulting nitrating species

Enitr, which we assumed to be a protein-bound ONOO2 species,

is formed in a fast step (reaction 7). The Enitr species can either

decay to E and NO32, self-nitrate the protein, or, in the pres-

ence of a bound phenolic substrate, lead to the formation of

O2N-PhOH (reaction 8), in competitive processes. ONOO2 is

able to perform phenol nitration in a single two-electron step,

without involving intermediate radical species.

Eþ NO2� $ E-NO2 (6)

E-NO2� þ H2O2 ! Enitr þ H2O (7)

Enitr þ PhOH $ ½Enitr-PhOH� !2HþEþ O2N-PhOHþ H2O (8)

Our studies show that both mechanisms, via NO2 and

ONOO2, can be operative in the nitration of phenolic com-

pounds, with a relative importance that depends on the enzyme

employed (e.g., lactoperoxidase or horseradish peroxidase) and

42 MONZANI ET AL.

on the NO22 concentration. At low [NO2

2], the reaction mainly

proceeds through NO2, while at high [NO22], the ONOO2-de-

pendent pathway prevails. The two competing pathways were

confirmed through the investigation of phenylacetic acid and 4-

hydroxybenzonitrile as mechanistic probes. Phenylacetic acid is

a good probe for ONOO2 because it reacts with ONOO2 gen-

erating nitrated and/or hydroxylated compounds, while it is

unreactive to NO2. The reactivity of 4-hydroxybenzonitrile is

totally different, because it easily reacts with NO2 generating 4-

hydroxy-3-nitrobenzonitrile, while ONOO2 is a poor nitrating

agent for this substrate (36).

The mechanistic results obtained from the study of phenol

nitration catalyzed by the peroxidase/NO22/H2O2 system can

be extended to the heme-protein/NO22/H2O2 systems, which

also bear physiological relevance. The met form of Mb and

Hb exhibits pseudo-peroxidase activity: it activates H2O2 fol-

lowing a catalytic cycle and generating active intermediates,

analogous to those of peroxidases (25). In the presence of

NO22 (besides H2O2), Mb, Hb, and also Ngb catalyze the

nitration of exogenous phenolic substrates through the forma-

tion of reactive species (NO2 and ONOO2) produced by the

peroxidase/NO22/H2O2 system (23, 24, 67, 68). Moreover, the

target of these reactions could be the endogenous Tyr and Cys

residues of the proteins. The physiological relevance of these

reactions raises from several aspects: (i) the presence of

nitrated proteins is often observed in pathophysiological condi-

tions (33, 69–71); (ii) the search of the biochemical pathways

responsible for biological nitration and the identification of

specific protein targets for nitration is essential for the under-

standing of the mechanisms of NO-derived pathologies (72);

(iii) during inflammatory diseases, the levels of H2O2 and

NO22 markedly increase (57, 58, 60, 73); and (iv) both Mb

and Hb are present in large amount in the body (5, 12), so that

even the low efficiency exhibited by their reactions, compared

with peroxidases, could be of physiological relevance; Ngb is

present in low concentration in the brain but reaches �100 lMin retinal cells (74, 75).

To investigate the endogenous modifications undergone by

Mb, Hb, and Ngb in the presence of NO22 and H2O2, the pro-

tein derivatives obtained upon reaction with various amounts of

NO22 and H2O2 (including [NO2

2] and [H2O2] close to the lev-

els that can be reached in vivo under conditions of oxidative

stress) were analyzed by HPLC-ESI-MS/MS. The main targets

of the oxidizing and nitrating species are the heme prosthetic

group and the Tyr and Cys residues. Modification of the cofac-

tor can be detected by direct HPLC-MS/MS analysis of the

acidified solution of the protein derivative, while the modifica-

tions at the amino acid residues were characterized in the poly-

peptide fragments resulting from tryptic digestion of the apo-

protein (apoMb, apoHb, or apoNgb) derivatives. The MS/MS

spectra of the modified peptide fragments were analyzed with

the SEQUEST1 algorithm, allowing the assignment of the mod-

ifications to the nitration of Tyr and the oxidation of Cys resi-

dues (23, 24, 67, 68).

THE hhMb/NO22/H2O2 SYSTEM: HEME AND Tyr

NITRATION

In vivo Mb may be present up to 0.2 mM within the cytosol

of myocardial cells and prevalently exists as oxyMb (12).

However, high concentrations of oxidants, such as those present

under inflammatory conditions, or acidic pH may induce autoxi-

dation of the protein with the formation of metMb. This is the

state in which Mb can exhibit a peroxidase like activity. The ki-

netic studies of the hhMb/NO22/H2O2 promoted nitration of dif-

ferent Tyr derivatives, the reduction of the protein intermediate

compound II (MbFeIV5O) by NO22 or the phenol, and the in-

hibition effect of NO22 at low [H2O2] indicate the existence of

the two competing paths (involving NO2 and a protein-bound

ONOO2 species) also responsible for the peroxidase-catalyzed

nitration. The occurrence of one or the other of these mecha-

nisms depends on NO22 concentration, as it was confirmed by

monitoring the predominant protein intermediate present in so-

lution during turnover. At low [NO22], the spectrum of the

MbFeIV5O intermediate, that is, the protein active species

accumulated during turnover according to the NO2 path, was

observed. At high [NO22], the spectrum of a different interme-

diate, that we suggested to be an iron-ONOO2 species

(MbFeIII-N(O)OO), was observed (23).

The nitrating species produced by the hhMb/NO22/H2O2 sys-

tem also react with endogenous Tyr residues and the heme pros-

thetic group [at one of the vinyl groups (76)]. The extent of

nitration of the cofactor, that is the major site of modification in

hhMb, depends on the amount of oxidant added to the reaction

mixture, and on the presence of an external phenol [such as 3-

(4-hydroxyphenyl)-propionic acid, HPA] (Table 1). Our results

indicate that phenolic substrates protect the protein from self-

nitration; in the absence of added phenols, and forcing the con-

ditions with high concentrations of the reactants, the nitration at

one of the two Tyr residues, Tyr146, was also observed, while

Tyr103 remains unaffected (m0-hhMb derivative in Table 1)

Table 1

Nitration of the heme and Tyr146 in the hhMb derivatives

(expressed in %), upon modification with NO22,

H2O2, and HPAa

Protein Reagent

Heme-NO2

(%)

Tyr146-NO2

(%)

m-hhMb [NO22] 5 0.8 M 33 n.d.

[H2O2] 5 0.1 mM

m0-hhMb [NO22] 5 0.8 M 50 6

[H2O2] 5 1 mM

m00-hhMb [NO22] 5 0.8 M 14 n.d.

[H2O2] 5 1 mM

[HPA] 5 1 mM

aData were obtained at pH 7.5 (200 mM phosphate buffer) and 25 8C. From (23).n.d., not detected.

43HEME-PROTEIN SELF-MODIFICATION

(23). Considering that the latter residue is exposed to solvent

and the former is an internal residue (Fig. 1), we can deduce

that the nitrating agent responsible for self-nitration of hhMb

does not come from the outside of the protein but the reaction

is intramolecular. Moreover, Tyr146 is located in proximity of

the Xe1 cavity of Mb (77, 78), and hence the nitrating species

produced close to or at the heme is free to diffuse to the Xe1

cavity and react with Tyr146.

The effect of the endogenous modifications (mainly heme

nitration) on the stability of hhMb to denaturation by guanidi-

nium chloride (Gdn-HCl) has been analyzed. The thermody-

namic parameters for unfolding and the Gdn-HCl concentration

causing 50% denaturation of hhMb and the m0-hhMb derivative

(obtained by the reaction of the protein with 0.8 M NO22 and

1.0 mM H2O2,) are reported in Table 2 (79). The similarity of

the unfolding parameters obtained with the native and modified

proteins indicate that the nitration of the heme and Tyr146 (50

and 6%, respectively) for the m0-hhMb derivative (Table 1)

have little effect on the stability of hhMb.

THE HMb/NO22/H2O2 SYSTEM: THE ROLE OF THE

Cys RESIDUE

Human Mb differs from the other known mammalian Mbs

for the presence of a cysteine residue at position 110. Like the

phenolic group of Tyr, also the reactive thiol group of Cys110

can be involved in reactions of pathological consequence that

could take place under oxidative stress and NO overexpression

in which the concentration of NO22 and H2O2 increases. In the

presence of these species, HMb as well as hhMb can perform

nitration of exogenous phenolic compounds. Although, of more

interest for the physiological consequences connected to the

source of the protein is the competitive endogenous derivatiza-

tion of HMb. Tandem mass analysis of HMb modified by

NO22 and H2O2 indicated the presence of two endogenous deri-

vatizations, besides nitration of the heme, that is, nitration of

Tyr residues (mainly Tyr103 and, to a lower extent, Tyr146),

and oxidation of Cys110 to sulfinic acid (Cys-SO2H) (Table 3

and Fig. 2) (24). Sulfinylation is not a rare event, given that 1–

2% of Cys residues of soluble proteins from rat liver were

detected as Cys sulfinic acid (80). Formation of sulfinic acid

can be reversed in the presence of specific proteins [such as sul-

firedoxin for Cys-SO2H of 2-Cys peroxiredoxins isoforms (81)],

but it might also be an irreversible process causing protein dam-

age, and this could be the case of HMb.

Of particular interest is the study of the modification of

HMb by reaction with NO22 and H2O2 in concentrations that

can occur in vivo, giving the m-HMb derivative. The presence

of both the sites of endogenous modification, that is, Tyr103

and Cys110, in the same tryptic peptide (Tyr103-Lys118)

allows speculating on the mechanism of the derivatization.

From HPLC-ESI-MS/MS analysis of the tryptic fragments from

apo-m0-HMb derivative, the modified Tyr103-Lys118 peptides

corresponding to the oxidation of Cys110 to sulfinic acid and to

both the oxidation of Cys110 and the nitration of Tyr103 were

found, while the peptide nitrated only at Tyr103 was absent.

Moreover, the Tyr103 residue is derivatized to a significantly

larger extent with respect to Tyr146 (Table 3), which is, never-

theless, the only one nitrated in hhMb, which lacks Cys (23,

24). All these results indicate that the nitration of Tyr103 is

mediated by the oxidation of the Cys residue through a free-rad-

ical pathway activated by NO2 (the predominant nitrating agent

at low [NO22]), according to Scheme 1.

The thiol group of HMb is oxidized first by NO2 to thiyl

radical, which in turn reacts with O2 to form a peroxy radical

(Cys110-SOO�). This radical produces the sulfinic acid after

hydrogen-atom abstraction from a reductant, according to path

Figure 1. Structure of hhMb showing the disposition of the side

chains of Tyr103 and Tyr146.

Table 2

Thermodynamic parameters for unfolding induced by Gdn-

HCl (DG0N�U and -m) and Gdn-HCl concentration causing

50% denaturation ([Gdn-HCl]0) of hhMb derivativesa

Protein

DG0N�U

(kcal mol21)

2m

(kcal mol21 M21)

[Gdn-HCl]0(M)

hhMb 5.91 4.50 1.318

m0-hhMb 5.37 4.01 1.345

aObtained by reaction of the protein with 0.8 M NO22 and 1.0 mM H2O2,

in 0.2 M phosphate buffer, pH 6.0, at 25 8C. From (79).

44 MONZANI ET AL.

a (Scheme 1); if the phenolic group of Tyr103 acts as the

reductant, the reaction of NO2 with the generated phenoxy radi-

cal results in the nitration of Tyr, according to path b (Scheme

1). The Tyr103-Lys118 peptide undergoes modification only at

Cys110 in the former case and at both Cys110 and Tyr103 in

the latter case. The residues Tyr103 and Cys110 are known to

display radical-type reactivity, for instance, the reaction of HMb

with H2O2, ONOO2, or NO2 leads to the formation of an inter-

molecular disulfide bond, nitration of Tyr103 and S-nitrosation

of C110, respectively (19, 31, 32, 82). The first step in the for-

mation of the protein homodimer is the oxidation of the Tyr103

residue to phenoxy radical (as a component of the compound I-

like species of HMb) (31, 32). In the presence of NO22, the

phenoxy radical is quenched by reaction with this anion and

NO2 is produced. Thus, NO2 reacts with the Cys thiol group,

and the subsequent oxidation of Tyr103 by the Cys peroxy

radical yields back the radical on the aromatic amino acid

(see Scheme 1). According to this scheme, Tyr103 nitration is

promoted by Cys110-SOO�, in agreement with the observation

that nitrated Tyr103 is present only together with Cys110-

SOOH.

The peculiar capability of the Cys residue in capturing the

oxidant species emerges also by comparing the data of heme

and Tyr nitration in hhMb with those in HMb derivatives

(Tables 1 and 3). In HMb, Cys110 protects the prosthetic group

from nitration. Tyr146 nitration, related to the fraction of the

nitrating species, which escapes from the active site and dif-

fuses to the Xe1 cavity, occurs to a similar extent in both the

proteins.

The catalytic activity of HMb modified by NO22 and H2O2

(m0-HMb derivative) in the nitration of phenolic compounds is

comparable to that of native HMb, because only a slight

decrease in the maximum turnover number (the kinetic parame-

ter kcat) can be noted for the modified protein (24). It follows

that the endogenous modifications (mainly oxidation of Cys110

and nitration of Tyr103) have little effect on the reactivity of

HMb intermediates; a similar negligible effect was obtained for

Figure 2. Structure of the Lys45Arg/Cys110Ala mutant of

HMb (78). The disposition of the side chains of Tyr103,

Cys110 (present in native HMb), and Tyr146 is shown.

Scheme 1. Pathways for the modification of cysteines and tyro-

sines residues in HMb.

Table 3

Nitration of the heme, Tyr103, and Tyr146, and oxidation of Cys110 in HMb derivatives (expressed in %), upon

modification with NO22 and H2O2

a

Modified protein Reagent Heme-NO2 (%) Tyr103-NO2 (%) Tyr146-NO2 (%) Cys110-SOOH (%)

m-HMb [NO22] 5 0.1 mM 0 44 0 76

[H2O2] 5 0.1 mM

m0-HMb [NO22] 5 0.8 M 8 54 10–20 95

[H2O2] 5 1 mM

aWith NO22 and H2O2, operating in 200 mM phosphate buffer, pH 7.5, at 20 8C. From (24).

45HEME-PROTEIN SELF-MODIFICATION

the nitration of the cofactor and of Tyr146 in hhMb on the sta-

bility of the protein to denaturation by Gdn-HCl (Table 1).

HUMAN HEMOGLOBIN SELF-MODIFICATIONINDUCED BY NO2

2 AND H2O2

It is now generally accepted that the human Hb could be

involved in many processes beside O2 transport. In particular,

metHb exhibits pseudo-peroxidase and nitrating activities in the

presence of H2O2 and NO22 which, being both able to diffuse

through the red blood cell membrane (83), can activate Hb in

vivo. Because the amount of Hb in the body can reach 2–5 mM

concentration, the side reactions promoted by Hb in the pres-

ence of NO22 and H2O2 may have physiological relevance. De-

spite the remarkable amount of work, it is still unclear if Hb

has a protective or damaging role towards RNS and reactive

oxygen species (ROS) produced by human body in response to

an inflammatory process.

H2O2 reacts with metHb to yield ferryl-Hb with a transient

radical localized on a protein residue, which after a one-electron

reduction forms the ferryl-Hb species; both of them have been

identified in human blood (84, 85). This implies that even if the

steady-state concentration of H2O2 in the blood does not exceed

1026 M (86) this is enough to cause metHb oxidation. The circu-

lating plasma NO22 concentration is instead between 0.1 and 1

lM; however, the local NO22 concentration is strictly correlated

with the extent of NO produced and can therefore significantly

increase during inflammatory processes (57). In the absence of

alternative substrates, in vitro treatment of metHb with NO22

and H2O2 has several consequences: nitration of the hemin group,

oxidation of Cys residues and nitration of Tyr residues strategi-

cally located in the protein and dimerization or even oligomeriza-

tion of the protein. The reaction, in fact, produces a complex

mixture of modified protein derivatives, as shown by RP-HPLC

analysis, and visual color change of the solution to greenish

brown. Besides, the peaks of the heme and the two subunits of

unreacted protein, the HPLC profile exhibits several unresolved

peaks for the modified protein chains and nitrated heme. Trypsin

digestion of the reacted protein followed by HPLC-ESI-MS/MS

analysis showed that four of the six Tyr residues of the protein

have been nitrated, and all the ��SH groups of the Cys residues

have been oxidized to sulfinic acids (Table 4).

In a recent work, Mouawad and coworkers (87) demon-

strated the existence of a wide cavity on the distal site of the a-Hb subunit. This cavity consists of three tunnels spreading from

the vicinity of the ligand-binding site to the surface of the subu-

nit, constituting possible passage ways for the entrance and the

exit of small ligands. Interestingly, the Tyr residues we found

to undergo nitration in the a-subunit, Tyr24 and Tyr42, are part

of the walls of one of these three tunnels. At the same time,

Tyr130 is part of one of the corresponding three tunnels in the

distal site of the b-subunit. According to Mouawad and cow-

orkers (87), the proximal site of both subunits is much more

compact than the distal site and only one tunnel seems to cross

the globin starting from the heme, leading to the surface. The

last Tyr residue we found to be nitrated, Tyr145 of the b-subu-nit, is exactly at the end of this tunnel. This data supports the

hypothesis that the nitrating agent is formed at the heme-bind-

ing site and reacts with the residues, which are on the way to

the protein surface.

Part of the complex RP-HPLC profile of the reaction prod-

ucts of Hb with NO22 and H2O2 is due to dimerization, and

possibly oligomerization of Hb. According to SDS-PAGE anal-

ysis in reducing conditions, dimerization occurs with high yield,

and the cross-link does not involve a disulfide bridge. As shown

by HPLC-ESI-MS/MS analysis of the dimer, the residues

involved in the covalent linkage are Tyr145 of the b-subunitand Tyr140 of the a-subunit. These two Tyr residues are located

at the end of the tunnel supposed by Mouawad and coworkers

(87) at the proximal site of the two subunits; therefore, they are

good targets for a radical reaction. Based on the crystal struc-

ture of native Hb, the Tyr residues seem too far to give rise to

a direct intramolecular reaction between the a- and the b-subu-nits of the same tetramer (Fig. 3). Therefore, it can be specu-

lated that the crosslinking occurs intermolecularly, that is,

between subunits of different Hb proteins. Although, it should

be noted that the structure of metHb is more flexible than that

of oxyHb, and the nitration reaction could cause further modifi-

cation in the protein structure, allowing an approach between

these two Tyr residues and hence an intramolecular coupling

process. This is in agreement with Edman analysis of the dimer,

which shows that it is composed of exactly 50% a- and 50% b-subunits. In addition, Western-blot analysis, employing a rabbit

polyclonal primary antibody directed against 3-O2N-Tyr, indi-

cates that both the individual subunits and the dimeric form of

Hb are nitrated.

di-Tyr formation has been observed in a number of patholo-

gies and is usually associated to oxidative stress conditions, as

in the formation of atherosclerotic plaques (88) or in Alzhei-

mer’s disease (89). The toxic effect of protein dimerization is

Table 4

Nitration of the heme group and Tyr residues, and oxidation

to sulfonic acid of Cys residues in metHb derivatives

(expressed in %), upon reaction with NO22 and H2O2

a

Modified residue %

Tyra24-NO2 10–20

Tyra42-NO2 30–40

Tyrb130-NO2 20–25

Tyrb145-NO2 40–50

Cysa104-SOOH 50–60

Cysb93-SOOH 70–80

Cysb112-SOOH 40–50

Heme-NO2 4–8

aThe reaction was performed on a sample of metHb (%1024 M) adding 0.1

M NaNO2 and 1 mM H2O2. From (67).

46 MONZANI ET AL.

well known because it is one of the severe side effects caused

by the administration of Hb-based blood substitutes (90). In

vivo dimerization of human Hb has not been reported so far,

but SDS-PAGE analysis, coupled with HPLC-ESI-MS analysis,

shows that a small amount of the Hb covalent dimer is also

present in the blood of healthy persons. The in vivo Hb dimeri-

zation, which could be connected to the same reaction we stud-

ied in vitro, may have several implications.

THE Ngb/NO22/H2O2 SYSTEM: ENDOGENOUS

MODIFICATIONS VIA ONOO2

Human Ngb is a recently discovered heme protein, expressed

in the nervous system with a yet unclear physiological function;

it belongs to the globin family but shares little homology with

Mbs and Hbs (74, 75, 91). Moreover, Ngb differs from the

other vertebrate globins also for the presence of a six-coordi-

nated heme (with His96 and His64 acting as the fifth/proximal

and sixth/distal ligands, respectively) in both the ferrous and

ferric forms (92, 93); as a result, the reactivity of the protein to-

ward exogenous ligands (such as NO, O2 or CO for the Ngb-

FeII form and NO22 for the Ngb-FeIII form) is limited by the

dissociation rate of the distal His residue (68, 92). The affinity

of Ngb for exogenous ligands depends also on the presence of

an internal disulfide bond between Cys46 and Cys55, which

affects the location of the distal His-containing helix, and whose

formation or cleavage is controlled by the redox state of the

cell (94, 95). As a consequence, the disulfide-bridged form of

the protein (NgbS��S) exhibits a higher affinity than the thiol

form (NgbSH) for small ligands such as O2 for ferrous (96) and

NO22 for ferric Ngb (68). A further peculiar property of Ngb is

that both NgbS��S and NgbSH are surprisingly able to perform

nitration of phenolic substrates in the presence of NO22 and

H2O2, with a reactivity only slightly lower than that of the anal-

ogous HMb/NO22/H2O2 system (68). This is very interesting

because the activation of alternative reactions by Ngb requires

the coordination of exogenous ligands in competition with the

distal His. The reaction here can only occur through the

ONOO2 pathway (involving the initial coordination of NO22 to

the iron center) (68). On the contrary, the NO2 pathway can be

excluded because the reactivity of Ngb with H2O2, and the con-

sequent generation of the protein ferryl intermediates is

extremely low (68, 97). The kinetic parameters for the phenol

nitration, the NO22-binding studies and the analysis of the

nitrated products from different substrates all support the hy-

pothesis that Ngb generates an active species with the chemical

properties of ONOO2.

The involvement of Ngb in the activation of NO22 and

H2O2 may have physiological relevance, because the metNgb

form is supposed to be generated in vivo by the scavenging ac-

tivity of toxic species under conditions of oxidative stress (91).

Our results indicate that, besides acting as a scavenger, Ngb can

be the source of RNS that react with protein residues, thus

affecting its physiological activity. In particular, we character-

ized the endogenous modifications undergone by Ngb (both in

the NgbS��S and NgbSH forms) in the presence of various

amount of NO22 and H2O2. Also in this case, the presence of

the internal S��S bond affects the functional properties of the

protein; the targets of the oxidizing and nitrating species

derived from the analysis of the MS/MS spectra are, besides the

heme prosthetic group, the protein tyrosine residues (Tyr44,

Tyr88, Tyr115, and Tyr137), which are transformed into 3-

O2N-Tyr, and Cys residues (Cys46, Cys55, and Cys120 in

NgbSH; only Cys120 in NgbS��S), which are oxidized to both

sulfinic (RSO2H) and sulfonic (RSO3H) acids (Fig. 4) (68).

Figure 3. View of the crystal structure of oxyHb. The heme

group, the residues Tyra24, Tyra42, Tyrb130, and Tyrb145,involved in nitration, and the residues Tyra140 and Tyrb145,involved in the subunits cross-link, are highlighted in black. Figure 4. Structure of the Cys46Gly/Cys55Ser/Cys120Ser mu-

tant of Ngb (117). The disposition of the side chains of Tyr44,

Tyr88, Tyr115, and Tyr137, and of Cys46, Cys55, and Cys120

present in the wild-type protein are shown. Heme proximal and

distal residues (His64 and His97, respectively) are also high-

lighted.

47HEME-PROTEIN SELF-MODIFICATION

Of particular interest is the analysis of the modifications

undergone by NgbSH, presumed to be the predominant form

in vivo (96), by reaction with low concentrations (i.e., close to

‘‘pathophysiological’’ values) of NO22 and H2O2 (Table 5). Cys

residues showed the highest reactivity towards the ONOO2

active species, and their reaction protects from nitration of Tyr

residues and the heme prosthetic group; nevertheless, the forma-

tion of sulfinic and sulfonic acids might be an irreversible pro-

cess causing protein damage. Actually, for Ngb, the oxidation

state of Cys46 and Cys55 is relevant because it is linked to the

peculiar six-coordination state of the heme; moreover, our stud-

ies confirm the importance of the internal S��S bond in control-

ling the reactivity of metNgb in the presence of NO22 and

H2O2.

Differently from the case of HMb, in which nitration of

Tyr103 is mediated by the oxidation of Cys110 through a free-

radical pathway activated by the reactive species NO2, Cys oxi-

dation and Tyr nitration in Ngb are independent. This is related

to the ONOO2-like active species, which is able to carry out

both oxidations through a free-radical pathway analogous to

that reported in Scheme 1 and also through single two-electron

oxidations.

The stability to unfolding in the presence of Gdn-HCl for

native and modified NgbSH shows that these proteins have con-

siderably higher stability than Mb. For instance, the [Gdn-HCl],

causing 50% denaturation of Ngbs and hhMb, are 4.1 M and

1.32 M, respectively. Moreover, the stability to denaturation of

the modified NgbSH is similar to that of the native protein indi-

cating that Tyr nitration and Cys oxidation have little effect on

the stability of the protein, as previously observed for the nitra-

tion of the heme and Tyr residues in hhMb (68).

HEME-PROTEIN MODIFICATION BY CATECHOLS

Biogenic catechols and, in particular, catecholamines, such

as the neurotransmitter dopamine and its precursor L-dopa,

are important molecules that can act as substrates in oxidative

processes promoted by proteins and enzymes in vivo. Cate-

chols can be easily oxidized by several oxidizing species to

the corresponding semiquinone radicals or to quinones and

then further react giving rise to polymeric products that may

end up with the formation of insoluble precipitates. These

reactions are responsible for the formation of neuromelanin in

the brain, particularly that of the substantia nigra pars com-

pacta and the locus coeruleus (98, 99). The quinone species

formed upon oxidation of dopamine are reactive species, indi-

cated as DAQ, which contribute to the oxidative stress in neu-

ral cells. Of particular importance is the modification induced

by DAQ on protein residues (41), through nucleophilic addi-

tion of polar groups of amino acids to the reactive conjugated

double bond of the quinone. DAQ-protein adducts have been

found for neuronal proteins such as a-synuclein (42), the

human dopamine transporter (43), tyrosine hydroxylase (44),

parkin (45), and many other proteins (46, 47) and enzymes

(44, 48–51). Interestingly, the reaction of the oxidative metab-

olite of dopamine with proteins has been considered to con-

tribute to neurodegeneration (41, 45, 100–102). The DAQ

modification of target proteins can apparently induce changes

in their structures and properties, aggregation, precipitation,

and, in some instances, promote cell death. In fact, DAQ-pro-

tein adduct formation has been shown to stabilize protofibril-

lar aggregates of a-synuclein, thus preventing the formation

of insoluble amyloidogenic fibrils (103, 104), and to induce

the formation of insoluble, SDS-stable, and large aggregates

of parkin (45).

The first reactive intermediate generated by one-electron ox-

idation of dopamine is its semiquinone radical. The reactivity

of this radical may be different from that of DAQ. Under oxi-

dative stress conditions, in which higher levels of H2O2 are

produced, activation of heme proteins and, in particular, peroxi-

dases can occur. The mechanism for the H2O2-dependent oxi-

dation of catecholic compounds by these enzymes has been

elucidated (105, 106). The pathway involves the formation of

semiquinone radicals as primary products, followed by dispro-

portionation to quinone and catechol. The same mechanism can

be followed by Mb, which is known to oxidize phenolic com-

pounds to radical species (107). As for peroxidases, both the

two protein intermediates generated by Mb upon reaction with

H2O2 can oxidize catechols to the corresponding radicals (108).

These reactive species can diffuse into the solution, in which

they can undergo a sequence of reactions leading to oligomeri-

zation (reactions 9–11):

(9)

Table 5

Nitration of Tyr88 and oxidation of Cys46, Cys55, and

Cys120 in the NgbSH derivative (all expressed in %), upon

reaction with NO22 and H2O2

2a

Modified residue %

Tyr88-NO2 1

Cys46-SOOH 2.3

Cys46-SO3H 4.5

Cys55-SOOH 2.5

Cys55- SO3H 7

Cys120-SOOH 10

Cys120-SO3H 9

aDerivative obtained upon reaction of the protein (6 3 1025 M) with NO22

(0.1 mM) and H2O22 (0.15 mM) in 200 mM phosphate buffer, pH 7.5.

From (68).

48 MONZANI ET AL.

In competitive processes, dopamine semiquinone and DAQ can

interact with amino-acid residues. The limited semiquinone re-

dox potential prevents its reaction with Tyr or Trp residues,

whereas the reaction with a Cys residue can produce a cysteinyl

radical (reaction 12):

The cysteinyl radical can be quenched by reducing agents in so-

lution or give rise to a coupling reaction with another radical,

that is, a semiquinone or a cysteinyl radical.

The DAQ species can undergo addition by a nucleophilic

compound present in solution (H��Nu), through a Michael-

type reaction, giving rise to a mixture of isomeric catechols

(reaction 13):

(13)

and after a second oxidation and addition, also undergo multiple

additions (109).

DAQ species react preferentially with Cys (110), but also

the imidazole group of His (111), the phenolic group of Tyr,

the terminal amino group, and the e-NH2 of Lys residues may

be involved (112).

To get insight into the sequential reactions occurring when

semiquinone is formed, we studied the oxidation of dopamine

by H2O2 catalyzed by metMb in the presence of N-acetyl-Cys.

The complex product mixture obtained has then been separated

by HPLC and characterized by MS and NMR. The four major

products formed are shown in Scheme 2 (unpublished data) in

which formation of 2-S-5-S-di-N-acetyl-Cys-dopamine, II, is

probably connected to a second addition of an N-acetyl-Cys to

the 5-S-N-acetyl-Cys-dopamine, I, after its further oxidation.

Interestingly, the same product pattern, with similar rela-

tive abundances is observed when the reaction is catalyzed

by peroxidases (data not shown), or performed electrochemi-

cally (113), indicating that the product distribution is ruled

by the reactivity of intermediates. Cystine, IV, formation

can be accounted for by the coupling between two cysteinyl

radicals.

The enzyme tyrosinase is very efficient in the oxidation of

L-dopa and dopamine to the corresponding quinones using

molecular O2 in a two-electron oxidation process (without for-

mation of the semiquinone species). When the oxidation of

dopamine by O2 is performed in the presence of Cys or N-

acetyl-Cys, the reaction produces a mixture of cysteinyl-dopa-

Scheme 2. The four major products formed upon oxidation of

dopamine by H2O2 catalyzed by metMb in the presence of N-

acetyl-cysteine.

49HEME-PROTEIN SELF-MODIFICATION

mine (Cys-DA) adducts resembling the distribution of species

generated by the heme proteins but with the lack of cystine.

Such a similar product composition for the Cys-DA adducts

formed by the two types of reactions, the one-electron oxida-

tion (catalyzed by heme proteins) and two-electron oxidation

(catalyzed by tyrosinase), indicates that the reaction is not

controlled by the semiquinone species. It means that the cou-

pling between semiquinone and cysteinyl radicals (reaction

14) does not occur but the former species is readily quenched

by Cys to the catechol form. Therefore, the product distribu-

tion is controlled by the reaction between quinone and Cys

(reaction 15) for both types of reactions. The hallmark of a

radical mechanism thus remains the formation of cystine

dimers (reaction 16).

(14)

Mb is a good target for the investigation of the capability of

the reactive species generated by catechols of modifying amino-

acid residues of the heme protein that generated them. In partic-

ular, HMb contains 20 Lys (residues 17, 35, 43, 46, 48, 51, 57,

63, 64, 78, 79, 80, 88, 97, 99, 103, 119, 134, 141, and 148), 9

His (residues 24, 36, 48, 64, 81, 82, 93, 97, and 119) and a sin-

gle Cys (110). Although the three-dimensional structure of

HMb has not been resolved yet, most probably the amino-acid

disposition does not differ significantly from that of the HMb

mutant Ala110Cys, the structure of which is known (114). This

structure allows to identify the amino acids in the protein inte-

rior or exposed to the solution. The modification of internal res-

idues indicates that either the active species diffuses inside the

protein or the modification occurs on the unfolded, or partially

unfolded, protein.

When HMb is incubated with dopamine in the presence of

H2O2, a modification of protein residues by the dopamine semi-

quinone and quinone formed by the catalytic activity of the pro-

tein is observed. Modification of Cys110 by a dopamine residue

(1151 amu) occurs to a significant extent (10–20%), depending

on the reaction conditions. Most probably, the Cys thiol group

reacts with DAQ forming the Cys-DA derivative. Interestingly,

Cys110 is not directly exposed to the protein surface, and its

modification probably involves partial protein unfolding. In

addition, while modification of HMb never occurred on Lys res-

idues, probably because at neutral pH the protonated Lys resi-

dues are not nucleophilic; in some cases, we observed that His

residues were modified.

With the aim of confirming the occurrence of modification

of His residues by dopamine and H2O2, also hhMb was treated

with the reagents and the tryptic peptides analyzed by HPLC-

ESI-MS/MS. The peptide containing the His81 and His82 with

a mass increase by a value corresponding to two dopamines res-

idues (1302 amu) was found, with a yield of 5–10%. The MS/

MS data are consistent both with the addition of two dopamine

residues to the above-mentioned His residues (Fig. 5) or with

the addition of a dopamine dimer to a single His.

The catalytic activity exhibited by HMb or hhMb with dopa-

mine/H2O2 occurs with concomitant melanin formation. During

the progress of the reaction, a relevant protein loss is observed.

The oligomers formed as intermediates in the process of mela-

nin formation are also reactive species and are able to react

with protein residues. When this happens, Mb is incorporated

into the precipitate of melanin. The impossibility to control

these side reactions and the precipitation of the protein masks

the complete detection of the His residues modified by DAQ

species.

COVALENT MODIFICATION, UNFOLDING ANDAGGREGATION OF HMb INDUCED BY REACTIVEFLUOROQUINONES

As shown earlier, HMb is a good target for enzymatically

generated reactive DAQ species. To obtain more precise infor-

mation on which amino acid residues are involved in the initial

protein adducts before oligomerization, it is necessary to

employ a substrate forming a quinone with lower and controlled

50 MONZANI ET AL.

reactivity with respect to dopamine. 4-Fluorocatechol is a good

candidate because the quinone generated by this compound can-

not give internal cyclization, and the electron withdrawing fluo-

rine atom increases the electrophilic character of the quinone. At

the same time, 4-fluorocatechol is a poor substrate for the HMb/

H2O2 system, and to circumvent this problem, fluoroquinone for-

mation was induced by the use of tyrosinase and 3-fluorophenol,

the reactivity of which was previously studied by our group

(115). By varying the concentrations of 3-fluorophenol and tyro-

sinase, it is possible to control the rate of the phenol polymeriza-

tion process and accumulate in solution mostly the fluoroquinone

or the dimer resulting from coupling of the quinone with 3-fluoro-

phenol, both species will be indicated here as fQ.

These fQ species can react both to produce further oligomers

or undergo a nucleophilic addition by protein amino acid resi-

dues, followed by HF elimination. As reported in Scheme 3, in

the case of HMb, His residues have been shown to be the most

prone to modification. The reaction, a simple Michael addition,

produces a catechol that in turn can be easily oxidized to quinone

by tyrosinase or by redox exchange with other quinone species.

Upon trypsin treatment of the modified HMb, followed by HPLC-

ESI-MS/MS analysis, the residues His24, His36, His48, His64

and His82 were detected as sites of derivatization (Fig. 6) with a

sizeable percent modification in the range between 25 and 90%,

depending on the specific residue.

According to the three-dimensional structure of the

Lys45Arg/Cys110Ala mutant of HMb (78), all modified His

residues are on the protein surface with the only exception of

the heme distal His64 that is not easily accessible. It is worth

noting that no derivatization was detected at the Cys110 resi-

due. At present, we can only speculate that Cys110 is not acces-

sible to fluoroquinones or that the fQ-modification of His64 in

the neighborhood prevents its reaction with fQ. Because of the

possibility to access to various residues, fQ modification of

HMb produces a family of protein derivatives having more

acidic pIs with respect to the native protein (Fig. 6a).

An important consequence of fQ derivatization is that HMb

becomes more easily unfolded due to a progressive loss in a-he-lix with a concomitant increase in random coil content. Unfold-

ing studies carried out in the presence of Gdn-HCl show, in

fact, that the free energy parameter DG0N�U progressively

decreases with the extent of derivatization from 5 kcal mol21

for the wild-type HMb to 2.3 kcal mol21 for the fQ-HMb deriv-

atives (Fig. 6b). The data also show a clear dependence of the

extent of fQ-HMb modification on the time course of the reac-

tion, involving an increase in the solvent-exposed surface area

of the protein. Moreover, the fQ-HMb derivatives tend to

undergo aggregation (Fig. 6c). Transmission electron micros-

copy experiments show the presence of structured bodies of

moderate size, probably corresponding to small oligomeric pro-

tofibrillar aggregates, but indicate a complete absence of fibrils.

CONCLUSIONS

Reactive species generated by heme proteins in their pseudo-

peroxidase activity show a preference to react with the protein

that generated them with respect to diffusion into the solution

and reaction with a different protein molecule. This may be

seen as a protecting effect of the protein when it acts as a cata-

lyst in ‘‘side reactions’’ such as those generating RNS or DAQ

species. It looks like that the globin-type heme proteins make a

suicide protection from these undesirable reactive species. It is

also of interest that HMb, hhMb, Hb, and Ngb undergo a differ-

ent pattern of modifications when they generate NO2 or

ONOO2. For example, when these reactive species are gener-

ated by exposition to pathophysiological concentrations of

NO22 and hydrogen peroxide, they are almost completely

quenched by HMb mostly through the oxidation of Cys110 to

sulfinic acid. With hhMb, lacking the Cys residue, its protecting

effect is lost and NO2 and ONOO2 can diffuse to the solution.

In Hb, the protecting effect of the protein seems to be exerted

by cross-link formation between the subunits. With Ngb, all

Cys residues are modification sites and are preferred with

respect to the Tyr side chains. Exposure to self-generated RNS

does not seem to alter to a large extent the stability to denatura-

tion of the proteins by Gdn-HCl. Probably, the a-helix rich glo-

bin fold is little disturbed by Tyr nitration or Cys oxidation. It

remains to be assessed whether the modified proteins acquire

new properties such as altered affinity, in their reduced forms,

towards O2, or different reactivity in their ‘‘side reactions.’’

Modification of the proteins through DAQ linkages has more

dramatic effects on their stability. For fQ-modified HMb, the

stability to Gdn-HCl denaturation is reduced to a half of that of

Figure 5. Structure of hhMb showing the disposition of the side

chains of His81 and His82.

51HEME-PROTEIN SELF-MODIFICATION

native HMb. Furthermore, DAQ modification induces protein

precipitation. Interestingly, DAQ strongly prefers the nucleo-

philic addition by a Cys residue, whereas fQ appears to react

preferentially with His residues. This may depend on the fact

that the latter quinone was exogenously produced by the action

of tyrosinase, but we could also take into account the different

chemical properties of the two quinones. The electron-rich

DAQ may prefer the reaction with a soft nucleophile as the

Scheme 3. Formation of fQ species and their reaction with protein His residues.

Figure 6. HMb modification induced by the system tyrosinase/3-fluorophenol/O2. (a) 2D-SDS/PAGE on hMb-fQ; (b) Gdn-HCl

denaturation curves for native HMb (l) and the hMb-fQ adduct (*) monitored by absorbance variation of the Soret band of the

protein. The inset shows the linear plots of DG0obs versus [Gdn-HCl] for the same samples; and (c) TEM experiment showing oligo-

meric protofibrillar aggregates in hMb-fQ.

52 MONZANI ET AL.

Cys-SH group, while the presence of the electron withdrawing

fluorine substituent in fQ, which increases its hardness, may

lead to the preference for the reaction with the harder imidazole

nucleophile of His residues. RNS and DAQ species can together

act on a target protein, giving rise to cumulative but unpredict-

able effects. For instance, it has been shown that the exposure

of tyrosine hydroxylase to dopamine gives rise to the inactiva-

tion but at the same time protects the protein from nitration by

ONOO2 and NO2 (116).

REFERENCES1. Moore, G. R., and Pettigrew, G. W. (1990) Cytochromes c, Evolu-

tionary, Structural and Physicochemical Aspects. Springer-Verlag,

Berlin, Germany.

2. Sono, M., Roach, M. P., Coulter, E. D., and Dawson, J. H. (1996)

Heme-containing oxygenases. Chem. Rev. 96, 2841–2887.

3. Dunford, H. B., and Stillmann, J. S. (1976) On the function and

mechanism of action of peroxidise. Coord. Chem. Rev. 19, 187–251.

4. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Nitric oxide:

physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43,

109–142.

5. Antonini, E., and Brunori, M. (1971) Hemoglobin and Myoglobin in

their Reactions with Ligands. North-Holland, Amsterdam, The Neth-

erlands.

6. Ferguson-Miller, S., and Babcock, G. T. (1996) Heme/copper termi-

nal oxidases. Chem. Rev. 96, 2889–2907.

7. Dunford, H. B. (1999) Heme Peroxidases. Wiley-VCH, New York, NY.

8. Bosshard, H. R., Anni, H., and Yonetani, T. (1991) In Peroxidases in

Chemistry and Biology, Vol. 2 (Everse, J., Everse, K. E., and

Grisham, M. B., eds.). p. 51, CRC Press, Boca Raton, FL.

9. Ortiz de Montellano, P. R. (1995). Cytochrome P450, 2nd ed. Plenum

Press, New York, NY.

10. Worrall, J. A. R., Diederix, R. E. M., Prudencio, M., Lowe, C. E.,

Ciofi-Baffoni, S., Ubbink, M., and Canters, G. W. (2005) The effects

of ligand exchange and mobility on the peroxidase activity of a bac-

terial cytochrome c upon unfolding. ChemBioChem 6, 747–758.

11. Diederix, R. E. M., Ubbink, M., and Canters, G. W. (2002) Peroxi-

dase activity as a tool for studying the folding of c-type cytochromes.

Biochemistry 41, 13067–13077.

12. Brunori, M. (2001) Nitric oxide, cytochrome-c-oxidase and myoglo-

bin. Trends Biochem. Sci. 26, 21–23.

13. Brunori, M. (2001) Nitric oxide moves myoglobin centre stage.

Trends Biochem. Sci. 26, 209–210.14. Herold, S. (1999) Kinetic and spectroscopic characterization of an in-

termediate peroxynitrite complex in the nitrogen monoxide induced

oxidation of oxyhemoglobin. FEBS Lett. 443, 81–84.

15. Herold, S., Exner, M., and Nauser, T. (2001) Kinetic and mechanistic

studies of the NO-mediated oxidation of oxymyoglobin and oxy-

hemoglobin. Biochemistry 40, 3385–3395.

16. Liu, X., Miller, M. J. S., Joshi, M. S., Sadowska-Krowicka, H., Clark,

D. A., and Lancaster, J. R., Jr. (1998) Diffusion-limited reaction of

free nitric oxide with erythrocytes. J. Biol. Chem. 273, 18709–18713.

17. Møller, J. K., and Skibsted, L. J. (2002) Nitric oxide and myoglobins.

Chem. Rev. 102, 1167–1178.

18. Herold, S., and Rock, G. (2003) Reactions of deoxy-, oxy-, and meth-

emoglobin with nitrogen monoxide. Mechanistic studies of the

S-nitrosothiol formation under different mixing conditions. J. Biol.

Chem. 278, 6623–6634.

19. Witting, P. K., Douglas, D. J., and Mauk, A. G. (2001) Reaction of

human myoglobin and nitric oxide. Heme iron or protein sulfhydryl

(S) nitrosation dependence on the absence or presence of oxygen.

J. Biol. Chem. 276, 3991–3998.

20. Jia, L., Bonaventura, C., Bonaventura, C., and Stamler, J. S. (1996)

S-nitrosohaemoglobin: a dynamic activity of blood involved in vascu-

lar control. Nature 380, 221–226.

21. Zhuang, Y., and Hogg, N. (2004) S-Nitrosohemoglobin: A biochemi-

cal perspective. Free Radic. Biol. Med. 36, 947–958.

22. Herold, S. (2004) Nitrotyrosine, dityrosine and nitrotryptophan forma-

tion from metmyoglobin, hydrogen peroxide and nitrite. Free Radic.

Biol. Med. 36, 565–579.

23. Nicolis, S., Monzani, E., Roncone, R., Gianelli, L., and Casella, L.

(2004) Myoglobin catalyzed exogenous and endogenous tyrosine

nitration by nitrite and hydrogen peroxide. Chem. Eur. J. 10, 2281–

2290.

24. Nicolis, S., Pennati, A., Perani, E., Monzani, E., Sanangelantoni, A.

M., and Casella, L. (2006) Easy oxidation and nitration of human

myoglobin by nitrite and hydrogen peroxide. Chem. Eur. J. 12, 749–

757.

25. Monzani, E., Alzuet, G., Casella, L., Redaelli, C., Bassani, C., Sanan-

gelantoni, A. M., Gullotti, M., De Gioia, L., Santagostini, L., and

Chillemi, F. (2000) Properties and reactivity of myoglobin reconsti-

tuted with chemically modified protohemin complexes. Biochemistry39, 9571–9582.

26. Pironti, V., Nicolis, S., Monzani, E., Colonna, S., and Casella, L.

(2004) Nitrite increases the enantioselectivity of sulfoxidation cata-

lyzed by myoglobin derivatives in the presence of hydrogen peroxide.

Tetrahedron 60, 8153–8160.

27. Ozaki, S.-I., Yang, H., Matsui, T., Goto, Y., and Watanabe, Y. (1999)

Asymmetric oxidation catalyzed by myoglobin mutants. Tetrahedron:Asymm. 10, 183–192.

28. Ozaki, S., Matsui, T., and Watanabe, Y. (1996) Conversion of myo-

globin into a highly stereospecific peroxygenase by the L29H/H64L

mutation. J. Am. Chem. Soc. 118, 9784–9785.

29. Ozaki, S., Matsui, T., and Watanabe, Y. (1997) Conversion of myo-

globin into a peroxygenase: a catalytic intermediate of sulfoxidation

and epoxidation by the F43H/H64L mutant. J. Am. Chem. Soc. 119,6666–6667.

30. Kato, S., Yang, H., Ueno, T., Ozaki, S., Philips, G. N., Fukuzumi,

S. M., and Watanabe, Y. (2002) Asymmetric sulfoxidation and amine

binding by H64D/V68A and H64D/V68S Mb: mechanistic insight

into the chiral discrimination step. J. Am. Chem. Soc. 124, 8506–

8507.

31. Witting, P. K., and Mauk, A. G. (2001) Reaction of human myoglo-

bin and H2O2. Electron transfer between tyrosine 103 phenoxyl radi-

cal and cysteine 110 yields a protein-thiyl radical. J. Biol. Chem.

276, 16540–16547.

32. Witting, P. K., Mauk, A. G., Douglas, D. J., and Stoker, R. (2001)

Reaction of human myoglobin and peroxynitrite: characterising bio-

markers for myoglobin-derived oxidative stress. Biochem. Biophys.Res. Commun. 286, 352–356.

33. Ischiropoulos, H. (1998) Biological tyrosine nitration: a pathophysio-

logical function of nitric oxide and reactive oxygen species. Arch.

Biochem. Biophys. 356, 1–11.

34. Burner, U., Furtmuller, P. G., Kettle, A. J., Koppenol, W. H., and

Obinger, C. (2000) Mechanism of reaction of myeloperoxidase with

nitrite. J. Biol. Chem. 275, 20597–20601.

35. Herold, S., and Rehmann, F.-J. K. (2003) Kinetics of the reactions of

nitrogen monoxide and nitrite with ferryl hemoglobin. Free Radic.

Biol. Med. 34, 531–545.

36. Monzani, E., Roncone, R., Casella, L., Galliano, M., and Koppenol,

W. H. (2004) Mechanistic insight into the peroxidase catalyzed nitra-

tion of tyrosine derivatives by nitrite and hydrogen peroxide. Eur. J.

Biochem. 271, 895–906.

37. Kilinc, K., Kilinc, A., Wolf, R. E., and Grisham, M. B. (2001) Myo-

globin-catalyzed tyrosine nitration: no need for peroxynitrite. Bio-chim. Biophys. Res. Commun. 285, 273–276.

53HEME-PROTEIN SELF-MODIFICATION

38. Herold, S., and Rehmann, F.-J. K. (2001) Kinetic and mechanistic

studies of the reactions of nitrogen monoxide and nitrite with ferryl

myoglobin. J. Biol. Inorg. Chem. 6, 543–555.

39. Casella, L., Monzani, E., Roncone, R., Nicolis, S., Sala, A., and De

Riso, A. (2002) Formation of reactive nitrogen species at biological

heme centers: a potential mechanism of nitric oxide-dependent toxic-

ity. Environ. Health Perspect. 110, 709–711.

40. Roncone, R., Barbieri, M., Monzani, E., and Casella, L. (2006) Reac-

tive nitrogen species of physiological relevance: mechanism of for-

mation and biological targets. Coord. Chem. Rev. 250, 1286–1293.

41. Stokes, A. H., Hastings, T. G., and Vrana, K. E. (1999) Cytotoxic

and genotoxic potential of dopamine. J. Neurosci. Res. 55, 659–665.

42. Conway, K. A., Rochet, J.-C., Bieganski, R. M., and Lansbury, P. T.

Jr. (2001) Kinetic stabilization of the a-synuclein protofibril by a

dopamine-a-synuclein adduct. Science 294, 1346–1349.

43. Whitehead, R. E., Ferrer, J. V., Javitch, J. A., and Justice, J. B.

(2001) Reaction of oxidized dopamine with endogenous cysteine resi-

dues in the human dopamine transporter. J. Neurochem. 76, 1242–1251.

44. Xu, Y., Stokes, A. H., Roskoski, R., Jr., and Vrana, K. E. (1998) Do-

pamine, in the presence of tyrosinase, covalently modifies and inacti-

vates tyrosine hydroxylase. J. Neurosci. Res. 54, 691–697.45. LaVoie, M. J., Ostaszewski, B. L., Weihofen, A., Schlossmacher, M.

G., and Selkoe, D. J. (2005) Dopamine covalently modifies and func-

tionally inactivates parkin. Nat. Med. 11, 1214–1221.

46. LaVoie, M. J., and Hastings, T. G. (1999) Dopamine quinone forma-

tion and protein modification associated with the striatal neurotoxicity

of methamphetamine: Evidence against a role for extracellular dopa-

mine. J. Neurosci. 19, 1484–1491.

47. Hastings, T. G., Lewis, D. A., and Zigmond, M. J. (1996) Role of

oxidation in the neurotoxic effects of intrastriatal dopamine injec-

tions. Proc. Natl. Acad. Sci. USA 93, 1956–1961.

48. Kuhn, D. M., Arthur, R.E., Jr., Thomas, D. M., and Elferink, L. A.

(1999) Tyrosine hydroxylase is inactivated by catechol-quinones and

converted to a redox-cycling quinoprotein: possible relevance to

Parkinson’s disease. J. Neurochem. 73, 1309–1317.49. Kuhn, D. M., and Arthur, R. Jr. (1998) Dopamine inactivates trypto-

phan hydroxylase and forms a redox-cycling quinoprotein: possible

endogenous toxin to serotonin neurons. J. Neurosci. 18, 7111–7117.

50. Berman, S. B., and Hastings, T. G. (1997) Inhibition of glutamate

transport in synaptosomes by dopamine oxidation and reactive oxygen

species. J. Neurochem. 69, 1185–1195.51. Berman, S. B., Zigmond, M. J., and Hastings, T. G. (1996) J Modifi-

cation of dopamine transporter function: Effect of reactive oxygen

species and dopamine. J. Neurochem. 67, 593–600.

52. Mohsenin, V. (1994) Human exposure to oxides of nitrogen at ambient

and supra-ambient concentrations. Toxicology 89, 301–312.

53. Stamler, J. S., and Feelisch, M. (1996) In Methods in Nitric OxideResearch (Feelisch, M., and Stamler, J. S., eds.). p. 19, Wiley, Chichester,

NY.

54. Beckman, J. S., and Koppenol, W. H. (1996) Nitric oxide, superox-

ide, and peroxynitrite: The good, the bad, and ugly. Am. J. Physiol.271, C1424–-C1437.

55. Stamler, J. S., Lamas, S., and Fang, F. C. (2001) Nitrosylation.

The prototypic redox-based signaling mechanism. Cell 106, 675–

683.

56. Kelm, M., and Yoshida, K. (1996) In Methods in Nitric OxideResearch (Feelisch, M., and Stamler, J. S., eds.). p. 47–58, Wiley,

Chichester.

57. Torre, D., Ferrario, G., Speranza, F., Orani, A., Fiori, G. P., and

Zeroli, C. (1996) Serum concentrations of nitrite in patients with

HIV-1 infection. J. Clin. Pathol. (Lond.) 49, 574–576.58. Hunt, J., Byrns, R. E., Ignarro, L. J., and Gaston, B. (1995) Con-

densed expirate nitrite as a home marker for acute asthma. Lancet

346, 1235–1236.

59. Eiserich, J. P., Cross, C. E., Jones, A. D., Halliwell, B., and van der

Vliet, A. (1996) Formation of nitrating and chlorinating species by

reaction of nitrite with hypochlorous acid. A novel mechanism for

nitric oxide-mediated protein modification. J. Biol. Chem. 271,

19199–19208.

60. van der Vliet, A., Eiserich, J. P., Halliwell, B., and Cross, C. E.

(1997) Formation of reactive nitrogen species during peroxidase-cata-

lyzed oxidation of nitrite. A potential additional mechanism of nitric

oxide-dependent toxicity. J. Biol. Chem. 272, 7617–7625.

61. Sampson, J. B., Ye, Y. Z., Rosen, H., and Beckman, J. S. (1998)

Myeloperoxidase and horseradish peroxidase catalyze tyrosine nitra-

tion in proteins from nitrite and hydrogen peroxide. Arch. Biochem.Biophys. 356, 207–213.

62. Eiserich, J. P., Hristova, M., Cross, C. E., Jones, A. D., Freeman, B.

A., Halliwell, B., and van der Vliet, A. (1998) Formation of nitric

oxide-derived inflammatory oxidants by myeloperoxidase in neutro-

phils. Nature 391, 393–397.

63. Shibata, H., Kono, Y., Yamashita, S., Sawa, Y., Ochiai, H., and

Tanaka, K. (1995) Degradation of chlorophyll by nitrogen dioxide

generated from nitrite by the peroxidase reaction. Biochim. Biophys.

Acta 1230, 45–50.

64. Lehnig, M. (2001) 15N chemically induced dynamic nuclear polariza-

tion during reaction of N-acetyl-L-tyrosine with the nitrating systems

nitrite/hydrogen peroxide/horseradish peroxidase and nitrite/hypo-

chloric acid. Arch. Biochem. Biophys. 393, 245–254.

65. Brennan, M. L., Wu, W., Fu, X., Shen, Z., Song, W., Frost, H., Vadseth,

C., Narine, L., Lenkiewicz, E., Borchers, M. T., Lusis, A. J., Lee, J. J.,

Lee, N. A., Abu-Soud, H. M., Ischiropoulos, H., and Hazen, S. L.

(2002) A tale of two controversies: defining both the role of peroxidases

in nitrotyrosine formation in vivo using eosinophil peroxidase and

myeloperoxidase-deficient mice, and the nature of peroxidase-generated

reactive nitrogen species. J. Biol. Chem. 277, 17415–17427.

66. Anni, H., and Yonetani, T. (1992) Degradation of environmental

pollutants by microorganisms and their metalloenzymes. Met. Ions

Biol. Syst. 28, 219–241.

67. Barbieri, M., Roncone, R., Monzani, E., Galliano, M., and Casella, L.

To be submitted to FASEB J.

68. Nicolis, S., Monzani, E., Ciaccio, C., Ascenzi, P., Moens, L., and

Casella, L. (2007) Biochem. J. 407, 89–99.

69. Xie, Q. W., and Nathan, C. (1994) The high-output nitric oxide path-

way: role and regulation. J. Leukocyte Biol. 56, 576–582.

70. Saleh, D., Barnes, P. J., and Giaid, A. (1997) Increased production of

the potent oxidant peroxynitrite in the lungs of patients with idiopathic

pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 155, 1763–1769.

71. Halliwell, B. (1997) What nitrates tyrosine? Is nitrotyrosine specific

as a biomarker of peroxynitrite formation in vivo?. FEBS Lett. 411,

157–160.

72. Turko, I. V., and Murad, F. (2002) Protein nitration in cardiovascular

diseases. Pharmacol. Rev. 54, 619–634.

73. Pryor, W. A., and Squadrito, G. L. (1995) The chemistry of peroxyni-

trite: a product from the reaction of nitric oxide with superoxide.

Am. J. Physiol. 268, L699–L722.

74. Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) A

vertebrate globin expressed in the brain. Nature 407, 520–523.

75. Schmidt, M., Giessl, A., Laufs, T., Hankeln, T., Wolfrum, U., and

Burmester, T. (2003) How does the eye breathe? Evidence for neuro-

globin-mediated oxygen supply in the mammalian retina. J. Biol. Chem.

278, 1932–1935.

76. Bondoc, L. L., and Timkovic, R. (1989) Structural characterization of

nitrimyoglobin. J. Biol. Chem. 264, 6134–6145.

77. Brunori, M., and Gibson, Q. H. (2001) Cavities and packing defects

in the structural dynamics of myoglobin. EMBO Rep. 8, 674–679.

78. Frauenfelder, H., McMahon, B. H., Austin, R. H., Chu, K., and

Groves, J. T. (2001) The role of structure, energy landscape, dynamics,

54 MONZANI ET AL.

and allostery in the enzymatic function of myoglobin. Proc. Natl. Acad.Sci. USA 98, 2370–2374.

79. Roncone, R., Monzani, E., Labo, S., Sanangelantoni, A. M., and

Casella, L. (2005) Catalytic activity, stability, unfolding, and degra-

dation pathways of engineered and reconstituted myoglobins. J. Biol.Inorg. Chem. 10, 11–24.

80. Hamann, M., Zhang, T., Hendrich, S., and Thomas, J. A. (2002)

Quantitation of protein sulfinic and sulfonic acid, irreversibly oxi-

dized protein cysteine sites in cellular proteins. Methods Enzymol.

348, 146–156.

81. Woo, H. A., Jeong, W., Chang, T.-S., Park, K. J., Park, S. J., Yang,

J. S., and Rhee, S. G. (2005) Reduction of cysteine sulfinic acid by

sulfiredoxin is specific to 2-Cys peroxiredoxins. J. Biol. Chem. 280,

3125–3128.

82. Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) Reaction of

human myoglobin and H2O2. Involvement of a thiyl radical produced

at cysteine 110. J. Biol. Chem. 275, 20391–20398.

83. Jensen, F. B., and Angisola, C. (2005) Perfusion of the isolated trout

heart coronary circulation with red blood cells: effects of oxygen

supply and nitrite on coronary flow and myocardial oxygen consump-

tion. J. Exp. Biol. 208, 3665–3674.

84. Giulivi, C., and Davies, J. A. (1994) Hydrogen peroxide-mediated

ferrylhemoglobin generation in vitro and in red blood cells. Methods

Enzymol. 231, 490–496.

85. Svistunenko, D. A., Patel, R. P., Voloshchenko, S. V., and Wilson,

M. T. (1997) The globin-based free radical of ferryl hemoglobin is

detected in normal human blood. J. Biol. Chem. 272, 7114–7121.

86. Chance, B., Sies, H., and Boveris, A. (1979) Hydroperoxide metabo-

lism in mammalian organs. Physiol. Rev. 59, 527–605.

87. Mouawad, L., Marechal, J.-D., and Perahia, D. (2005) Internal cav-

ities and ligand passageways in human hemoglobin characterized by

molecular dynamics simulations. Biochem. Biophys. Acta 1724, 385–393.

88. Leeuwenburgh, C., Rasmussen, J. E., Hsu, F. F., Mueller, D. M.,

Pennathur, S., and Heinecke, J. W. (1997) Mass spectrometric quanti-

fication of markers for protein oxidation by tyrosyl radical, copper,

and hydroxyl radical in low density lipoprotein isolated from human

atherosclerotic plaques. J. Biol. Chem. 272, 3520–3526.

89. Hensley, K., Maidt, M. L., Yu, Z. Q., Sang, H., Markesbery, W. R.,

and Floyd, R. A. (1998) Electrochemical analysis of protein nitrotyro-

sine and dityrosine in the Alzheimer brain indicates region-specific

accumulation. J. Neurosci. 18, 8126–8132.

90. Everse, J., and Hsia, N. (1997) The Toxicities of Native and Modified

Hemoglobins. Free Radic. Biol. Med. 22, 1075–1099.

91. Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp, M.,

Laufs, T. L., Roesner, A., Schmidt, M., Weich, B., Wystub, S.,

Saaler-Reinhardt, S., Reuss, S., Bolognesi, M., De Sanctis, D., Mar-

den, M. C., Kiger, L., Moens, L., Dewilde, S., Nevo, E., Avivi, A.,

Weber, R. E., Fago, A., and Burmester, T. (2005) Neuroglobin and

cytoglobin in search of their role in the vertebrate globin family.

J. Inorg. Biochem. 99, 110–119.

92. Trent, J. T. III, Watts, R. A., and Hargrove, M. S. (2001) Human

neuroglobin, a hexacoordinate hemoglobin that reversibly binds oxy-

gen. J. Biol. Chem. 276, 30106–30110.

93. Uno, T., Ryu, D., Tsutsumi, H., Tomisugi, Y., Ishikawa, Y., Wilkin-

son, A. J., Sato, H., and Hayashi, T. (2004) Residues in the distal

heme pocket of neuroglobin. Implications for the multiple ligand

binding steps. J. Biol. Chem. 279, 5886–5893.

94. Hamdane, D., Kiger, L., Dewilde, S., Green, B. N., Pesce, A., Uzan,

J., Burmester, T., Hankeln, T., Bolognesi, M., Moens, L., and

Marden, M. C. (2004) Coupling of the heme and an internal disulfide

bond in human neuroglobin. Micron 35, 59–62.

95. Hamdane, D., Kiger, L., Dewilde, S., Green, B. N., Pesce, A., Uzan,

J., Burmester, T., Hankeln, T., Bolognesi, M., Moens, L., and Mar-

den, M. C. (2003) The redox state of the cell regulates the ligand

binding affinity of human neuroglobin and cytoglobin. J. Biol. Chem.278, 51713–51721.

96. Fago, A., Hundahl, C., Dewilde, S., Gilany, K., Moens, L., and

Weber, R. E. (2004) Allosteric regulation and temperature depend-

ence of oxygen binding in human neuroglobin and cytoglobin.

Molecular mechanisms and physiological significance. J. Biol. Chem.

279, 44417–44426.

97. Herold, S., Fago, A., Weber, R. E., Dewilde, S., and Moens, L.

(2004) Reactivity studies of the Fe(III) and Fe(II)NO forms of human

neuroglobin reveal a potential role against oxidative stress. J. Biol.

Chem. 279, 22841–22847.

98. Zecca, L., Zucca, F. A., Wilms, H., and Sulzer, D. (2003) Neurome-

lanin of the substantia nigra: a neuronal black hole with protective

and toxic characteristics. Trends Neurosci. 26, 578–580.

99. Sulzer, D., Bogulavsky, J., Larsen, K. E., Behr, G., Karatekin, E.,

Kleinman, M. H., Turro, N., Krantz, D., Edwards, R. H., Greene, L.

A., and Zecca, L. (2000) Neuromelanin biosynthesis is driven by

excess cytosolic catecholamines not accumulated by synaptic

vesicles. Proc. Natl. Acad. Sci. USA 97, 11869–11874.

100. Hastings, T. G., and Berman, S. B. (1999) In Role of Catechol Qui-

none Species in Cellular Toxicity (Creveling, C. R., ed.). p. 69–89,

F. P. Graham Publishing, Johnson City, TN.

101. Xu, J., Kao, S. Y., Lee, F. J., Song, W., Jin, L. W., and Yankner, B.

A. (2002) Dopamine-dependent neurotoxicity of a-synuclein: a mech-

anism for selective neurodegeneration in Parkinson disease. Nat.

Med. 8, 600–606.

102. Dong, Z., Ferger, B., Paterna, J. C., Vogel, D., Furler, S., Osinde,

M., Feldon, J., and Bueler, H. (2003) Dopamine-dependent neurode-

generation in rats induced by viral vector-mediated overexpression of

the parkin target protein, CDCrel-1. Proc. Natl. Acad. Sci. USA 100,

12438–12443.

103. Li, J., Zhu, M., Manning-Bog, A. B., Di Monte, D., and Fink, A. L.

(2004) Dopamine and L-dopa disaggregate amyloid fibrils: implica-

tions for Parkinson’s and Alzheimer’s disease. FASEB J. 18, 962–964.

104. Cappai, R., Leck, S.-L., Tew, D. J., Williamson, N. A., Smith, D. P.,

Galatis, D., Sharples, R. A., Curtain, C. C., Ali, F. E., Cherny, R. A.,

Culvenor, J. G., Bottomley, S. P., Masters, C. L., Barnham, K. J.,

and Hill, A. F. (2005) Dopamine promotes a-synuclein aggregation

into SDS-resistant soluble oligomers via a distinct folding pathway.

FASEB J. 19, 1377–1379.

105. Metodiewa, D., Rezka, K., and Dunford. H. B. (1989) Oxidation of

the substituted catechols dihydroxyphenylalanine methyl ester and

trihydroxyphenylalanine by lactoperoxidase and its compounds. Arch.

Biochem Biopsys. 274, 601–608.

106. Ferrari, R. P., Laurenti, E., Ghibaudi, E. M., and Casella, L. (1997)

Tyrosinase-catecholic substrates in vitro model: kinetic studies on the

o-quinone/o-semiquinone radical formation. J. Inorg. Biochem. 68,

61–69.

107. Redaelli, C., Monzani, E., Santagostini, L., Casella, L., Sanangelantoni,

A. M., Pierattelli, R., and Banci, L. (2002) Characterization and Peroxi-

dase Activity of a Myoglobin Mutant Containing a Distal Arginine.

ChemBioChem 3, 226–233.

108. Hayashi, T., Hitomi, Y., Ando, T., Mizutani, T., Hisaeda, Y., Kita-

gawa, S., and Ogosh, H. (1999) Peroxidase activity of myoglobin is

enhanced by chemical mutation of heme-propionates. J. Am. Chem.

Soc. 121, 7747–7750.

109. Tse, D. C. S., McCreery, R. L., and Adams, R. N. (1976) Potential

oxidative pathways of brain catecholamines. J. Med. Chem. 19, 37–40.

110. Xu, R., Huang, X., Morgan, T. D., Prakash, O., Kramer, K. J., and

Hawley, M. D. (1996) Characterization of products from the reactions

of N-acetyldopamine quinone with N-acetylhistidine. Arch. Biochem.

Biophys. 329, 56–64.

111. Huang, X., Xu, R., Hawley, M. D., and Kramer, K. J. (1997) Model

insect cuticle sclerotization: reactions of catecholamine quinones with

55HEME-PROTEIN SELF-MODIFICATION

the nitrogen-centered nucleophiles imidazole and N-acetylhistidine.Bioorg. Chem. 25, 179–202.

112. Schaefer, J., Kramer, K. J., Garbov, J. R., Jacob, G. S., Stejskal, E.

O., Hopkins, T. L., and Speirs, R. D. (1987) Aromatic cross-links in

insect cuticle: detection by solid-state 13C and 15N NMR. Science

235, 1200–1204.

113. Huang, X., Xu, R., Hawley, D., Hopkins, T. L., and Kramer, K. J.

(1998) Electrochemical oxidation of N-acyldopamines and regioselec-

tive reactions of their quinones with N-acetylcysteine and thiourea.

Arch. Biochem. Biophys. 352, 19–30.

114. Hubbard, S. R., Hendrickson, W. A., Lambright, D. G., and Boxer, S.

G. (1990) X-ray crystal structure of a recombinant human myoglobin

mutant at 2.8 A resolution. J. Mol. Biol. 213, 215–218.

115. Battaini, G., Monzani, E., Casella, L., Lonardi, E., Tepper, A. W.

J. W., Canters, G. W., and Bubacco, L. (2002) Tyrosinase-cata-

lyzed oxidation of fluorophenols. J. Biol. Chem. 277, 44606–

44612.

116. Park, S., Geddes, T. J., Javitch, J. A., and Kuhn, D. M. (2003)

Dopamine prevents nitration of tyrosine hydroxylase by peroxyni-

trite and nitrogen dioxide. Is nitrotyrosine formation an early

step in dopamine neuronal damage? J. Biol. Chem. 278, 28736–

28742.

117. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln,

T., Burmester, T., and Bolognesi, M. (2003) Human brain neuroglo-

bin structure reveals a distinct mode of controlling oxygen affinity.

Structure 11, 1087–1095.

56 MONZANI ET AL.