doi:10.1016/j.jmb.2006.02.042 J. Mol. Biol. (2006) 358, 1081–1093

A Rearranging Ligand Enables Allosteric Controlof Catalytic Activity in Copper-containingNitrite Reductase

Hein J. Wijma1, Iain MacPherson2, Maxime Alexandre1

Rutger E. M. Diederix1, Gerard W. Canters1*, Michael E. P. Murphy2

and Martin Ph. Verbeet1

1Leiden Institute of ChemistryLeiden University, P.O. Box9502, 2300 RA Leiden, TheNetherlands

2Departments of Biochemistryand Molecular Biology and ofMicrobiology and ImmunologyUniversity of British ColumbiaVancouver, BC V6T 1Z3Canada

0022-2836/$ - see front matter q 2006 E

Present addresses: M. Alexandre,Biophysics, Faculty of Sciences, VrijHV Amsterdam, The Netherlands; RDepartamento de Biofisica, Instituto“Rocasolano” CSIC, Serrano 119, 28

Abbreviations used: wt, wild-typreductase; DMS, dimethylsulfide; Nelectrode.

E-mail address of the correspondcanters@chem.leidenuniv.nl

In Cu-containing nitrite reductase from Alcaligenes faecalis S-6 the axialmethionine ligand of the type-1 site was replaced (M150G) to make thecopper ion accessible to external ligands that might affect the enzyme’scatalytic activity. The type-1 site optical spectrum of M150G (A460/A600Z0.71) differs significantly from that of the native nitrite reductase (A460/A600Z1.3). The midpoint potential of the type-1 site of nitrite reductaseM150G (EMZ312(G5) mV versus hydrogen) is higher than that of the nativeenzyme (EMZ213(G5) mV). M150G has a lower catalytic activity (kcatZ133(G6) sK1) than the wild-type nitrite reductase (kcatZ416(G10) sK1). Thebinding of external ligands to M150G restores spectral properties, midpointpotential (EM!225 mV), and catalytic activity (kcatZ374(G28) sK1). Alsothe M150H (A460/A600Z7.7, EMZ104(G5) mV, kcatZ0.099(G0.006) sK1)and M150T (A460/A600Z0.085, EMZ340(G5) mV, kcatZ126(G2) sK1)variants were characterized. Crystal structures show that the ligands actas allosteric effectors by displacing Met62, which moves to bind to the Cu inthe position emptied by the M150G mutation. The reconstituted type-1 sitehas an otherwise unaltered geometry. The observation that removal of anendogenous ligand can introduce allosteric control in a redox enzymesuggests potential for structural and functional flexibility of copper-containing redox sites.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: nitrate reductase; crystal structure; ligand; catalytic activity;copper

*Corresponding author

Introduction

Nature often uses copper to mediate electrontransfer in biological redox chains. For this purposethe copper is incorporated in a protein scaffold in amononuclear so-called type-1 site or in a closelyrelated dinuclear CuA site.1 These sites can be foundthroughout the kingdoms of life, from archaea to

lsevier Ltd. All rights reserve

Department ofe Universiteit, 1081. E. M. Diederix,Quımica Fısica

006, Madrid, Spain.e; NiR, nitriteHE, normal hydrogen

ing author:

humans. In photosynthesis and respiration smalltype-1 site containing proteins (cupredoxins) shuttleelectrons between larger enzymes. In coppercontaining enzymes, type-1 (or CuA) sites enableelectron transfer between catalytic sites and externalelectron donors. The enzymes are often involved inrespiration (nitrite reductase, cytochrome c oxidase)or in the conversion of metabolites (multi-copperoxidases). Some of them have found industrialapplication (laccase). Here, we show that proteinengineering can introduce allosteric control over thetype-1 site of copper-containing nitrite reductase(NiR) from Alcaligenes faecalis S-6.

The physiological role of NiR is the dissimilatoryreduction of nitrite ðNOK

2 C2HCCeK/NOCH2OÞ2

although NiR does catalyze bidirectionally; at pH 8the kcat of the reverse reaction is higher than the kcat ofthe forward reaction.3 NiR is a homotrimer, in whicheach subunit contains a type-1 copper site that

d.

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M150TM150H

(a)

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Figure 1. Optical spectra of native and mutant nitritereductases. (a) NiR wt and NiR M150G. (b) NiR M150Hand M150T. Notice the different vertical scale in bothpanels. Buffer: 20 mM Mops (pH 7.0), TZ298 K, proteinconcentrations around 50 mM. For further experimentaldetails see Materials and Methods.

1082 Allosteric Control by a Rearranged Ligand

transfers electrons from a physiological electrondonor to a type-2 copper site that is located deeperinside the enzyme.4–6 The type-2 copper forms theactive site together with a water network and anAsp-His pair, that binds the nitrite and donatesprotons.7–10

In a type-1 site, two histidine residues and onecysteine bind the copper; these three ligands arevery strongly conserved. In addition, one or twoweaker binding, axial ligands can be present.A methionine or a glutamine can serve as thefourth (axial) ligand and sometimes a fifth axialligand, in the form of a backbone carbonyl oxygenfrom glycine, can bind on the opposite side.11 Thetwo-histidine/one-cysteine ligand set results inunique spectroscopic properties of the oxidizedtype-1 site (Cu2C; the Cu1C state is spectroscopi-cally silent). All characterized type-1 copper siteshave a unique small hyperfine splitting in theirelectron paramagnetic resonance spectrum.11 Fur-thermore, strong absorption bands at approxi-mately 600 nm and often also around 460 nmresult in a blue or green color, depending mostlyon the binding geometry of the weaker axialligands. Here, we will refer to these absorptionbands as 460 and 600 nm bands also when they areslightly shifted.

One approach to study the function of a ligand ina metal site is to engineer the ligand out and addexternal ligands that may bind in the gap created inthe first coordination shell.12 The interesting ques-tion is then how the binding of the external ligandaffects the properties of the metal site. Earlier thisapproach was used to investigate the type-1 coppersite in azurin.12–19 By using the enzyme nitritereductase, it is possible to monitor the electrontransfer function, which is connected with thecatalytic activity. For NiR, we earlier found20 thatwhen this approach was applied to the C-terminalhistidine ligand, catalytic activity was lost becausethe midpoint potential of the type-1 site was alteredtoo much, also in the presence of external ligands.Because axial ligands less drastically influence themidpoint potential of the type-1 site than theequatorial ligands,11,21–28 we investigated whetherin such an axial cavity variant the electron transferfunction could be better restored by externalligands. This question was not addressed in earlierreports.18,29,30

We replaced the original type-1 methionineligand in NiR by a glycine (M150G) to createspace for an external ligand such as an imidazoleor an alcohol. As controls for these ligands weprepared the M150H and M150T variants. Theoptical absorption of the T1-site was used tomonitor the binding of exogenous ligands.The absorption of the type-2 site (e790nmZ100 MK1 cmK1)20 is so weak that it does notinterfere with this. External ligands were found tobind near the engineered type-1 site and to restorethe midpoint potential of this site and the catalyticactivity of the enzyme. Crystal structures show thatthe external ligands act as allosteric effectors. Their

binding causes a surprising structural rearrange-ment by which Met62 becomes part of the type-1copper-site.

Results

Spectral characterization and binding ofexternal ligands

Purified NiR M150G is blue, unlike wild-type(wt) NiR, which is green. A UV/Vis spectrum(Figure 1(a)) shows that the blue color is theresult of a change in the relative contributions ofthe absorption bands around 460 and 600 nm (NiRwt: e460Z2900 MK1 cmK1, e589Z2200 MK1 cmK1;NiR M150G: e457Z2000 MK1 cmK1, e600Z2800 MK1 cmK1). Two additional mutants wereproduced as controls, one for “strong axial inter-action” (imidazole side-chain in M150H) and onefor “weak axial interaction” (alcohol side-chain inM150T). For NiR M150H and NiR M150T the visiblespectrum did differ significantly from the wt NiRspectrum (Figure 1(b)). In NiR M150H the 460 nmband has gained intensity and is shifted tosignificantly shorter wavelengths, while a weak

Table 1. Affinity constants of allosteric effectors for thetype-1 site of NiR M150G

External ligand KoxD (mM)

Dimethylsulfide 21G4Ethylmethylsulfide 14G6Formamide 172G40

Allosteric Control by a Rearranged Ligand 1083

absorption is visible at 547 nm (e439Z4600 MK1 cmK1, e547Z600 MK1 cmK1). For M150Talmost all absorption is present in the 600 nmband (e460Z400 MK1 cmK1, e602Z4700 MK1 cmK1).As a result NiR M150H is yellow and NiR M150Tis blue.

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(a)

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(c)

dimethylsulfide

propanol

imidazole

pyridine

acetonitrile

formamide

Figure 2. Titration of NiR M150G with external ligands.(a) Effect of acetamide on the optical spectrum of NiRM150G. Arrows indicate the direction of the spectralchanges occurring upon subsequent additions of acet-amide. (b) The absorption at 600 (open triangles) and460 nm (filled circles) plotted versus acetamide concen-tration. The lines are fits based on a model correspondingwith a single binding site as described in Materials andMethods. (c) Optical spectra, shifted vertically withrespect to each other, of NiR M150G in the presence ofvarious external ligands. The concentrations were:dimethylsulfide, 151 mM (Z7!KD); propanol, 1940 mM(Z5.5!KD); imidazole, 250 mM (Z5!KD); pyridine,30 mM (Z11.5!KD); acetonitrile 1036 mM (Z14.5!KD);formamide 850 mM (Z5!KD).

Acetamide 71G3Imidazole 52G3Ethanol 805G142Propanol 353G52Acetonitrile 71.5G7.3Pyridine 2.6G0.24-Methylthiazole 22G3Nitrite 157G27

All the ligands in this Table displayed isosbestic points duringtitration of the type-1 spectrum. For details see Materials andMethods.

To study ligand binding to the oxidized type-1site of M150G the optical spectrum was monitoredupon addition of different compounds (Figure 2(a)).A variety of ligands all caused a stronger absorptionat 460 nm, and a weaker absorption at 600 nm.Isosbestic points were observed, and the titrationdata could be fit to a single binding site (Figure 2(b);Table 1). Although the used compounds includedpotentially strong axial ligands (e.g. imidazole/acetamide), weak axial ligands (e.g. propanol),and ligands of similar strength as a methionine(e.g. dimethylsulfide), all resulting spectra weresimilar (Figure 2(c)) and reminiscent of wt NiR(Figure 1(a)). Ratios of A460 over A600 were about thesame: for example for acetamide-saturated NiRM150G e458Z2900, e600Z1800 MK1 cmK1, andA460/A600Z1.6 (for wt A460/A600Z1.3) and quitedifferent from M150H (A460/A600Z7.7) and M150T(A460/A600Z0.085). The only exception was thespectrum resulting from the titration with forma-mide, for which the 460 nm peak shifted to asignificantly shorter wavelength and the peak at360 nm was less intense (Figure 2(c), bottom trace).The remarkable similarity among the spectraobserved with all the other ligands suggests thatthey trigger a similar change in the ligand sphere atthe type-1 copper site, without directly binding tothe copper.

For imidazole bound M150G, a further change ofthe optical spectrum was observed on a longer time-scale (Figure 3). After 6 h the visible spectrum wasstable, the 460 band had shifted to a significantlyshorter wavelength (431 nm) and the A460/A600 ratiohad increased (Figure 3). When the imidazole wasremoved by dialysis, the original spectrum of M150Gwithout ligands was observed (results not shown).For other ligands (acetamide, formamide, DMS) notime dependence of the spectrum was observed overa period of weeks at room temperature.

Midpoint potential

Midpoint potentials were determined to definethe driving force for electron transfer. The midpointpotential of the type-1 site of wt NiR was found to

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time (min)0 60 120 180 240 300 360

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30 n

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Figure 3. The optical spectrum of NiR M150G-imidazole versus time. Spectra of M150G were recordedevery 10 min in the presence of imidazole (260 mMZ5!KD) at 25 8C. Arrows indicate the direction of the spectralchange. The inset shows the absorption at 430 nm versustime. The continuous line in the inset is a fit to a singleexponential (yielding a rate of 0.823(G0.003) hK1).

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Figure 5. Midpoint potential of NiR versus ligandconcentration. (a) Midpoint potential of NiR M150G(open circles) and NiR wt (filled circles) versus acetamideconcentration. The thick gray line indicates the midpointpotential of wt NiR without ligand. The continuous line isa fit of the midpoint potential of M150G to equation (1).(b) Midpoint potential of NiR M150G and NiR wt versuspyridine concentration, legend as in (a).

1084 Allosteric Control by a Rearranged Ligand

be 213(G5) mV versus the normal hydrogenelectrode (NHE) (Figure 4). For NiR M150H themidpoint potential was extremely low with104(G5) mV. The midpoint potentials of M150T(340(G5) mV) and M150G without ligands(312(G5) mV) were higher than that of the wt NiR.

The variation in the midpoint potential of NiRM150G was measured as a function of the ligandconcentration for acetamide and pyridine (Figure 5).At high ligand concentrations the midpoint poten-tial of the wt NiR is also affected (Figure 5).Apparently, non-specific effects come into playcausing an increase in midpoint potential of wtNiR, which counteracts the decrease in midpoint

Potential versus SHE (mV)

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Figure 4. Redox-titrations of NiR variants. Downwardpointing triangles denote reductive titrations, upwardpointing triangles depict oxidative titrations. Filledtriangles indicate M150H and M150G, open triangleswild-type NiR and NiR M150T (as indicated in thegraph). The continuous lines are fits to the Nernstequation for the combined titrations.

potential of NiR M150G. Therefore, the valueobtained for the midpoint potential with ligandbound (EL

M, see equations (1) and (2)) was inter-preted as an upper-limit. With acetamide bound toNiR M150G the fitted midpoint potential was !225 mV versus NHE, and the Kox

D was 157(G13) mM.For pyridine bound NiR M150G the midpointpotential was !245 mV, and the Kox

D was 3.0(G0.5) mM. Thus, the midpoint potential of the type-1site with ligand did not differ substantially fromthat of the wt NiR.

Activity

The type-1 site of nitrite reductase is essential forcatalytic activity; thus, the electron transfer functionof type-1 site variants can be assessed by compari-son of the catalytic activity of the enzyme variantwith that of the wt NiR. Catalytic activity wasmeasured with the physiological electron donorpseudoazurin (Table 2). NiR M150H had fourorders of magnitude less activity than the wt NiR.

Table 2. Catalytic activity of NiR variants

NiR variantSaturated withexternal ligand ksat

cat (sK1) KappD (mM)

WT – 416G10 –M150H – 0.099G0.006 –M150T – 126G2 –M150G – 133G6 –M150G Dimethylsulfide 373G22 34G7M150G Ethylmethylsul-

fideO292 O73

M150G Formamide O255 O4000M150G Acetamide 374G28 1068G295M150G Acetonitrile O390a ND

For details of the activity measurements see Materials andMethods.

a Also the native NiR increased in activity (see Figure 6(b)).A lower limit means that it was not possible to saturate theactivity of NiR M150G with ligand. ND, not determined.

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Figure 6. Effect of external ligands on catalytic activity.Wt NiR, open circles; NiR M150G, triangles; the gray lineis a visual reference to the catalytic activity of native NiRin the absence of external ligands. (a) Rate of catalyticturnover versus dimethylsulfide concentration. The con-tinuous line is the least-squares fit that yielded theapparent dissociation constant and the maximum activity(see Materials and Methods for details). (b) Activity versusacetonitrile concentration.

Allosteric Control by a Rearranged Ligand 1085

The catalytic activities of NiR M150T and NiRM150G without ligands were one third of that ofthe wt NiR.

The activity of NiR M150G could be increased bythe addition of exogenous ligands (Figure 6).Acetamide and dimethylsulfide (DMS) restoredthe activity up to the level of wt NiR. Thedependence of activity versus ligand concentrationcould be fit to a one ligand binding equilibrium. Theresulting apparent dissociation constant was in allcases higher than the KD for binding to the oxidizedtype-1 site (Tables 1 and 2). For ethylmethylsulfideand formamide it was not possible to saturate NiRM150G with ligand; however, activity doubled overa concentration range in which the wt NiR hadconstant activity (Table 2).

The effects were less straightforward for othercompounds because they also influenced theactivity of wt NiR. In the case of acetonitrile(Figure 6(b)), the wt NiR catalytic activity increased50% in contrast to an increase of 200% for M150G.Wt and M150G NiR both decreased in activity withimidazole as a ligand. It is noteworthy thatstructurally different compounds restored theactivity to a level not significantly different fromthat of the wt NiR (Table 2).

Structure

Superposition of wt NiR to the DMS andacetamide-bound structures of M150G revealsthat these small molecules displace the side-chain of Met62, a residue near the type-1 coppersite that is non-coordinating in the wt structure(Figure 7). The remarkable finding is that in itsnew position Met62 adopts a conformation thatallows its Sd atom to take up a new position thatis similar to the Met150 Sd position in the nativewt structure. DMS and acetamide bind M150GNiR at nearly the same position, roughly 6 Afrom the protein surface. The DMS sulfur is0.46 A from the Sd atom of Met62 in wild-typeNiR. The DMS is in an orientation analogous to

that of the Met62 thioether that it displaces andtoo far from the Cu-atom (4.5 A) to be a ligand(Figure 7). Acetamide is slightly further awayfrom the Cu-atom (5.0 A) and forms hydrogenbonds to two buried water molecules. Theacetamide N and O atoms could not beunambiguously assigned. The two buried watermolecules are located in a 5 A deep tunnel thatconnects to the surface and also is present in thewt and M150G-DMS structures.

The displacement by DMS and acetamide of theMet62 side-chain is accomplished by a 1158 rotationof the c1 torsional angle, a 258 rotation of c2, and a598 rotation of c3. The atomic positions of the Met62backbone shift only slightly (0.03 A rms), but the ftorsional angle rotates 278. As a result of all torsionalchanges, the Met62 sulfur moves 4.5 A to bind to thetype-1 copper at a position that overlaps that of theMet150 Sd in wt NiR (Figure 7). The geometries ofthe type-1 sites are almost identical to that of wt NiR(Table 3). No other structural perturbations wereobserved surrounding the type-1 copper site.

Figure 7. Crystal structures of the type-1 copper sites. Identical views are given for (a), (b) and (c). Foreground: Ala61–Phe64, His95. Background: Cys136, Trp144, His145, andGly150 (mutant) or Met150 (wt). The type-1 copper is a salmon colored sphere. The sA weighted 2FoKFc electron density maps are contoured at 1s. (a) The structure of M150G-DMS. (b) The structure of M150G-acetamide. (c) Stereo stick representation of wt NiR superimposed with M150G-DMS and M150G-acetamide. The carbon atoms are coloredwhite, slate, and green for wt NiR, M150G-DMS, and M150G-acetamide, respectively.

Table 3. Metal ligand geometry of type-1 sites in wt nitritereductase and in M150G

Native AfNiR M150G DMSM150G

acetamide

Distances (A)Axial–Cu 2.48 2.39 2.3795–Cu 2.07 2.13 2.13136–Cu 2.22 2.21 2.24145–Cu 2.06 2.07 2.04Cu–NSNa 0.64 0.57 0.63Angles (deg.)136–Cu–95 129 129 126136–Cu–Axial

106 97 100

Axial–Cu–95 89 95 95Axial–Cu–145

133 130 129

qb 64 67 71

The numbers 95, 136, and 145 in the left column refer to the Nd ofHis95, the Sg of Cys136, and the Nd of His145. Axial refers to theSd of Met150 in the wt NiR (1SJM) and the Sd of Met62 in theM150G structures. Sigma values (standard deviations deter-mined from average values of three monomers in the asymmetricunit) amount to less than 5% for bond angles and less than 3% forbond distances.

a This is the distance between the Cu atom and the NSN planedetermined by the ligand atoms of residues His95/Cys136/His145.

b q is the dihedral angle between the planes through 136–Cu–Axial ligand and the plane through 95–Cu–145.

Allosteric Control by a Rearranged Ligand 1087

A second acetamide molecule is modeled in theactive site solvent channel, 7.3 A from the type-2copper. In the DMS-bound structure, additionaldensity is present at the substrate binding site of thetype-2 copper. This density is modeled as water butmay be DMS or a degradation product.

Discussion

Axial ligand binding and spectroscopy

In the crystal structures, the external ligandsdimethylsulfide and acetamide do not bind to theCu atom, but instead they displace Met62, which iscoordinated to the type-1 copper. The crystals aregrown below pH 5 and data were collected at liquidnitrogen temperature, so a different conformationcould prevail in solution at pH 7. This possibilitycould be excluded, however, by optical spec-troscopy.

For type-1 copper sites, the absorbance band at600 nm originates from p overlap between thecopper dx2-y2 and the sulfur orbitals, the 460 nmband originates from pseudo-s overlap between thesame orbitals.11 The A460/A600 ratios in blue copperproteins reveal variations in these overlaps. In thecase of a trigonal site, such as in azurins, the dx2-y2

orbital overlaps almost solely with the two histidineresidues and the cysteine, resulting in almost purep overlap with the cysteine. In tetrahedrallydistorted type-1 sites like in the nitrite reductaseof Alcaligenes faecalis S-6,4,31 the d-orbital overlaps

with the axial methionine (stronger axial inter-action). This change of orientation produces anincreased A460/A600 ratio,32,33 and a shift to shorterwavelengths11 of both absorption bands. Thechange in orientation of the dx2-y2 orbital can bequantified by the dihedral angle q between theplanes through 136–Cu–Axial and the planethrough 95–Cu–145 (see Table 3).32

The effects of strong versus weak axial interactionon the optical spectrum of a type-1 copper site canbe seen in two examples: NiR M150H (strong) andM150T (weak). The optical spectrum of M150H hasa very high ratio of A460/A600, and peaks that areshifted to shorter wavelengths, while M150T has avery low A460/A600 ratio. For NiR M150G aspurified the A460/A600 ratio is closer to that of thewt NiR than to M150T.31,32,34

Crystallography of the NiR M150G variantindicates that Met62 and not the added exogenousligands (DMS/acetamide) bind to the type 1 copper,and optical spectroscopy confirms the crystallo-graphic result. Binding of dimethylsulfide andethylmethylsulfide to NiR M150G restores thespectroscopic properties of wt NiR, which isexpected either when these thioether compoundsbind directly or, alternatively, when Met62 binds tothe Cu atom. For acetonitrile, ordinary alcohols(which mimic threonine), imidazoles (which mimichistidine) and acetamide (which mimics glutamine)essentially the same spectra are observed as withdimethylsulfide and ethylmethylsulfide. This resultis incompatible with direct binding of these groupsto the Cu-center and rather points to similarCu-sites in all these experiments. Crystallographicobservations on DMS and acetamide-soaked crys-tals of NiR M150G correlate well to the solutionoptical properties. Not only were the ligand-soakedcrystals green, also the q dihedral angles found inthe crystal structures, which correlate with theA460/A600 ratios, are similar for the wt and the twoM150G-ligand structures (Table 3). All these resultsindicate that the bound compounds affect theCu-site structure in the same indirect manner bycausing Met62 to bind to the Cu. Thus, theadded compounds may be considered as allostericeffectors.

Long-term incubation of NiR M150G withimidazole resulted in spectra indicative of adifferent axial ligand. A 30 nm peak shift to shorterwavelengths, accompanied by an increase in theA460/A600 ratio, suggests stronger axial interactionpossibly due to direct copper coordination byimidazole, similar to NiR M150H. Incubation withformamide significantly shifted the A460 peak also,indicating that formamide may bind directly, atleast partially, to the type-1 copper. Thus, someexogenous ligands may substitute for Met150 bycoordinating to the copper.

Midpoint potential and catalytic activity

The M150T mutation changed the midpointpotential by C127 mV with respect to the wt

Met62

S

NCu

N

S

Met62

S

NCu

N

S

Met62

S

NCu

N

S

Met62

S

NCu

N

S

T state R state

T-Ligand State R-Ligand State

M150cavity

Ligand

Ligand

Keq

Ligand

Ligand

M62cavity

Scheme 1. Allosteric control of nitrite reductaseM150G. The two nitrogen atoms and one sulfur atomthat coordinate the copper represent His95, His145, andCys136. The side-chain of Met62 is indicated. Thepositions of empty cavities are represented by filledspheres, those of ligand-filled cavities are shown bybroken circles. Met62 cavity refers to the space left after itsrearrangement with respect to the native structure;Met150 cavity denotes the cavity created by deletion ofthis residue.

1088 Allosteric Control by a Rearranged Ligand

protein, which resembles the shift of C107 mVobserved for Rhodobacter sphaeroides NiR M182T.26

For NiR M150G, the change in midpoint potential(C99 mV) resembles the change observed forAlcaligenes xylosoxidans NiR M144A (C74 mV35)and azurin M121A (C63 mV27). For NiR M150H,the shift in midpoint potential (K109 mV) is similarto the shift of K100 mV for Alcaligenes denitrificansazurin M121H.36 The observed variations in themidpoint potential are in line with the idea thatstronger axial interaction lowers the midpointpotential of the type-1 site.11 The higher midpointpotential of NiR M150T and NiR M150G withrespect to the wt may partly explain the lowercatalytic activity, since it will hinder the electrontransfer to the type-2 site. In A. xylosoxidans NiRM144A, the electron transfer rate from the type-1 totype-2 Cu site is indeed tenfold decreased.37 InAchromobacter cycloclastes NiR M150Q (changeK127 mV), the electron transfer rate from pseudoa-zurin to NiR had decreased below the detectionlimit,23 which is reminiscent of the low activity ofour NiR M150H (change K109 mV). Thus, in aqualitative sense the catalytic activities of our NiRvariants vary in agreement with the changes inmidpoint potential of the type-1 site.

To determine the midpoint potential of NiRM150G with an allosteric effector bound, we triedto saturate both the oxidized and reduced type-1sites with ligand (otherwise an average midpointpotential with and without ligand bound ismeasured according to equation (1)). Assuming asimple scheme, the Kox

D obtained from potentio-metric titration should be identical to that obtainedfrom direct ligand titration, which for pyridine isindeed the case. The calculated EM!225 mV versusNHE with acetamide as the allosteric ligand is notsignificantly different from the value for the wt NiR(213 mV versus NHE). It is unlikely that themidpoint potential is much lower than 225 mV,since the catalytic activity (which is a measure of theelectron transfer function) with acetamide as theligand is not distinguishable from that of the wt NiR(Table 2). Thus, binding of Met62 to the copperapparently restores the midpoint potential of thetype-1 site to the wt value and restores electrontransfer function as well.

Allosteric control

The results presented so far can be summarizedby Scheme 1. The states at the right and left-handcorners at the top of the square depict the type-1 sitein the absence of external ligands with Met62 in itsnative conformation and in a conformation where itbinds to the Cu, respectively. In the left confor-mation the cavity created by the Met150Glymutation is empty, in the right conformation it isoccupied by Met62 and a new cavity is created atthe original position of Met62. The two states at thebottom represent similar conformations but withthe cavities filled with a “ligand”. The states atthe left side of the square are denoted by “T”

(from “Tense” denoting an enzymatically less activestate),53 those at the right are denoted by “R”(from “Relaxed” denoting an enzymatically moreactive state).

The crystallographic results constitute clearevidence for the existence of the R-ligand state.As for the R-state, one may expect its opticalspectrum to be identical to that of the R-ligandstate, since the spectrum appears insensitive towhat is present in the Met62 cavity as long asMet62 is coordinated to the Cu. Since the spectrumof the Met150Gly variant in the absence of externalligands is different from when ligand is present,one may conclude that in the former species theMet62 is not coordinated to the Cu. This species isrepresented by the T-state (top left in Scheme 1).Crystals of the protein in this state could notbe obtained so far, since components of thecrystallization buffer tended to penetrate theprotein and to produce an R-ligand state.

The slow conversion of the R-ligand state in thepresence of imidazole into a new state with astrongly differing optical spectrum is an indicationfor the occurrence of a T-ligand state. Definite prooffor the occurrence of this state must await theoutcome of further crystallographic experiments,however, as well as further studies of the enzymaticactivity of this species. The simplest explanation

Allosteric Control by a Rearranged Ligand 1089

for the initial formation of an R-ligand state withimidazole is that the “Met62 cavity”, which has atunnel to the surface, is more accessible than theMet150 cavity, while the subsequent formation ofthe T-ligand state is much slower but thermodyna-mically more favorable.

The occurrence of the R-state at this stage ishypothetical; its actual occurrence according toScheme 1 depends on the values of the variousequilibrium constants and the ligand concentration.The important observation in the present context isthat conversion of the T-state (top left) with lowactivity into an R-ligand state with high activity canbe effected by adding a ligand to the solution. Thisis in contrast with earlier work showing thatreplacement of an (equatorial) ligand by a glycineresults in a protein that is inactive even in thepresence of external ligands.13,14,20

The difference between KappD and Kox

D (Tables 1and 2) we ascribe to binding of the allosteric effectorwith lower affinity to the reduced type-1 site(Figure 5). Under the turnover conditions in whichthe K

appD is measured, the type-1 site needs to accept

electrons from pseudoazurin and donate them tothe type-2 site. If the reduced type-1 site needs tobind the external ligand for efficient electrontransfer to the type-2 site, the K

appD will be a

weighted average between KoxD and Kred

D .The only two ligands (imidazole, and formamide)

that seem capable of providing a T-ligand state aresimilar in that both are expected to bind Cu(II) withhigher affinity than a thioether group.38 Conversely,alcohols are expected to bind weaker to Cu(II) thana thioether group, and indeed do not bind to the Cuof either azurin M121G or M121A.29 This obser-vation suggests that some of the ligands like ethanoldo not bind to the type-1 copper in NiR M150Gbecause the thioether group of the Met62 hasgreater affinity for Cu(II). When the ligand hashigher affinity for the cavity left by Met62, than forCu(II), then the R-state is also favored over theT-state.

In conclusion, the replacement of the axialmethionine in the type-1 site of NiR (Met150) bya glycine creates a protein variant of which theactivity can be restored to wt values by allostericeffectors. The presence of a nearby methionine(Met62) that can subsititue for Met 150 is crucialfor this to occur. As this methionine is conservedin many blue copper proteins39,40 the conversionof the wt form into a variant that can beactivated allosterically appears more generallyapplicable.

Materials and Methods

Materials

For mutagenesis and expression of NiR a pET28b-basedvector7 containing nirK from A. faecalis S-641 was used.For introduction of the mutations the following primerswere used together with standard molecular biology

techniques; standard forward primer, CAT GGT GCTGCC GCGGGA GGG TCT GCA TGA CG; M150G reverseprimer, GCC GTC ATG CAG ACC CTC CCG CGG CAGCAC CAT GAT CGC ACC ATT CCC GCC CGA TACGAC (underlined is a SacII restriction site that wasintroduced by a silent mutation; in bold are the alteredbases of which CCC is the antisense codon for Gly; forM150H the alteration was GTG, for M150T it was CGT).Expression and purification of NiR, and of its physio-logical electron donor pseudoazurin,42–44 were achievedas described.3,20

For crystallography and for activity assays a gel-filtration step was added as the last step of thepurification of NiR as described.3 The Cu-contentdetermined with bicinchoninic acid45 was 1.9 for wtNiR, 1.7 for NiR M150T, 1.7 for NiR M150H (quotednumbers are per monomer), and 1.0 for pseudoazurin.For NiR M150G the Cu-content varied between 1.7–2.1per batch; a batch with a Cu-content of 1.9 was used forthe activity assays and for crystallization.

Spectroscopy and assays

The spectrophotometer was a Perkin Elmer Instru-ments Lambda 800. Prior to measuring spectra, sampleswere centrifuged at 16,000g for 10 min to remove smallquantities (!5%) of aggregated protein that in the case ofNiR can produce a scattering contribution comparable inintensity to the absorption spectrum of the type-1 site.NiR M150G (50 mM) was titrated with ligands in 50 mMMops (pH 7.0). After correction for dilution both theincrease of absorbance (A) at 460 nm, and the decrease at600 nm were least-squares fitted assuming a singlebinding site (AZANoLigandCDA!½L�=ðKox

D C ½L�Þ, inwhich L is the free ligand concentration). For all theassays in the presence of ligands, the total ligandconcentration exceeded the protein concentration atleast tenfold and is therefore taken as equal to the freeligand concentration.

Activity assays were carried out by monitoring theoxidation of pseudoazurin as described.3 The concen-trations of the electron donor pseudoazurin (275–325 mM)and the electron acceptor nitrite (5 mM) were saturating.The concentration of NiR was typically 1 nM. The bufferfor activity assays was always 50 mM Mops (pH 7.0).Whenever using volatile compounds, the cuvette wassealed with a PTFE stopper. All reported activities werecalculated from initial rates. Apparent dissociationconstants ðK

appD Þ were obtained from a least-squares fit

of activity (v) versus ligand concentration tovZvNoLigandCDv!½L�=ðK

appD C ½L�Þ. The meaning of

KappD is explained in Discussion.

Potentiometric titrations

Potentiometric titrations were carried out as describedby Dutton46 in a cuvette held at 298 K in 100 mMpotassium phosphate (pH 7.0). The NiR concentrationwas typically 40 mM. Diaminodurol (2,3,5,6-tetramethyl-1,4-phenylenediamine) was used as a redox mediator at100–200 mM. Potassium ferricyanide and sodium dithio-nite were used to change the potential of the solution.Visible absorption and the potential of the solution weremonitored until both were stable. Spectra were recordedin the range of 510–800 nm since diaminodurol givesnegligible absorbance in this region. For the M150Hmutant, phenazine methosulfate (N-methyldibenzo-pyrazine methyl sulfate, 10 mM) was used as a redox

Table 4. Crystallographic data collection and refinementstatistics

CrystalM150G

DimethylsulfideM150G Aceta-

mide

Cell dimensions (A) aZ61.97 aZ61.40bZ103.0 bZ102.4cZ146.0 cZ146.3

Resolution (A) 1.80 (1.85–1.80)a 1.60 (1.64–1.60)r-merge 0.068 (0.292) 0.098 (0.320){I}/{s(I)}b 22.1 (6.43) 10.8 (3.15)Completeness (%) 86.5 (93.8) 83.0 (82.0)Unique reflections 76,078 (8146) 100,959 (9877)Working R-factor 0.166 0.177Free R-factor 0.199 0.209rmsd bond length (A) 0.009 0.008Overall B-factor (A2)c 19.8 25.9Water molecules 1165 1158

a Values in parenthesis are for the highest resolution shell.b {I}/{s(I)} is the average intensity divided by the average

estimated error in intensity.c B-factors are an average from all three monomers.

1090 Allosteric Control by a Rearranged Ligand

mediator while the scan range was 400–800 nm.The absorption of oxidized NiR M150H (30 mM) exceededthat of the phenazine methosulfate tenfold.

The recorded spectra were integrated using a routinewritten in Igor Pro (WaveMetrics Inc., see SupplementaryData for the routine code). For baseline correction thisroutine approximated the scattering contributions (due toaggregated protein) either by a linear approximation orby a method described elsewhere.47 There was no need tocorrect for the type-2 site contribution since the absorp-tion of the type-2 site in this part of the spectrum is 30times lower than that of the type-1 site.20 The integratedabsorbance versus potential was fitted to the Nernstequation with the number of electrons held at one.Therefore, the midpoint potential versus ligand concen-tration was fitted to equation (1):48

EM ZENLM KðRT=FÞln½Kred

D !ðKoxD C ½L�Þ=ðKox

D !ðKredD

C ½L�ÞÞ� (1)

in which ENLM is the midpoint potential without ligand, [L]

denotes the free ligand concentration, KoxD and Kred

D are theligand dissociation constants from the oxidized andreduced type-1 site, respectively, R is the gas constant, F isthe Faraday constant and T is the absolute temperature.Because the ligand concentration far exceeded the proteinconcentration, [L] was set equal to the total ligandconcentration. The midpoint potential of the type-1 sitewith the external ligand bound ðEL

MÞ was calculated fromequation (2):

ELM ZENL

M KðRT=FÞln½KredD =Kox

D � (2)

A series of control experiments46 were carried out toexclude artifacts due to the binding of a redox mediator,oxidant or reductant to the protein. The midpoint potentialsof M150G and wt NiR were also determined in the absenceof diaminodurol using higher concentrations of ferro/ferricyanide (1–10 mM) as redox mediator, which gaveidentical results. Replacing sodium dithionite withL-ascorbic acid gave an identical midpoint potential forthe wt NiR, but slower equilibration. When TMPD andDCPIP were used as redox-mediators, as has been done forNiR from Rhodobacter sphaeroides,26 identical results wereobtained. However, we preferred not to use the latter twomediators since they absorb in the same spectral region asnitrite reductase, and resulted in a slower equilibration. Forevery midpoint potential here reported, both a reductiveand an oxidative titration were carried out. They resulted inthe same midpoint potentials. We could not detectsignificant differences in the midpoint potential of fullyCu-loaded M150G (2.0 Cu per monomer) and partiallytype-2 depleted batches (1.7 Cu per monomer). Thepotential of the reference electrode was calibrated withquinhydrone (0.2 g in 10 ml of 100 mM phosphate buffer(pH 7.0) gives a solution potential of 286 mV versus theNHE).

Structure determination

Met150Gly crystals were grown at room temperatureby the hanging-drop vapor-diffusion method. The crystal-lization conditions were 10 mM sodium acetate (pH 4.5),2 mM zinc acetate, 2 mM cupric sulfate, 60–100 mMammonium sulfate, and 4–10% poly(ethylene glycol)6000. A stock protein concentration of 35 mg/ml in

10 mM Tris (pH 7) was used. These conditions resultedin blue crystals that grew in an orthorhombic lattice(space group P212121). Once grown, crystals were soakedin mother liquor containing either 2 mM DMS or 200 mMacetamide until they turned from blue to greenindicating an alteration of the type-1 copper site.Crystals were then transferred to mother liquorsupplemented with glycerol as a cryoprotectant andeither DMS or acetamide. DMS-soaked crystals werelooped into a cryostream (Oxford Cryo Systems) forhome source diffraction studies using a MAR345detector and Rigaku RU-300 X-ray generator. Aceta-mide-soaked crystals were looped and immersed inliquid nitrogen for data collection using a MAR345detector at the Stanford Synchrotron Radiation Labora-tory (beamline 7-2). Both DMS and acetamide-soakedcrystals diffracted to greater than 1.8 A resolution anddiffraction data were processed with DENZO.49

DMS and acetamide-soaked M150G crystals containthe NiR trimer in the asymmetric unit. A 1.4 A resolutionstructure of nitrite-soaked wt NiR50 was used as thestarting refinement model after removal of the Met150side-chain, nitrite and selected water molecules. Thestructures were refined using REFMAC51 with 5–7% ofthe data set aside for calculation of the free R-factor.FoKFc difference maps were used to locate the acetamideand DMS ligands and to define the conformation ofMet62. The copper ligand geometry and positions of thecopper atoms were not restrained throughout therefinement. Each chain of both structures begins atAla4 and ends at Gly339. At least 90% of the residues ineach structure occupy the most favorable position in theRamachandran plot as described by PROCHECK.52

Statistics of data processing and structure refinementare presented in Table 4.

Protein Data Bank accession codes

The atomic coordinates have been deposited withthe RCSB Protein Data Bank and are availableunder accession codes 1ZDQ (M150G-DMS) and 1ZDS(M150G-Acm).

Allosteric Control by a Rearranged Ligand 1091

Acknowledgements

This research was funded in part by andNSERC Discovery Grant to M.E.P.M. and I.S.M.was supported by an NSERC graduate student-ship. Portions of this research were carried out atthe Stanford Synchrotron Radiation Laboratory(SSRL), a national user facility operated byStanford University on behalf of the US Depart-ment of Energy, Office of Basic Energy Sciences.The SSRL Structural Molecular Biology Programis supported by the Department of Energy, Officeof Biological and Environmental Research, by theNIH National Center for Research Resources,Biomedical Technology Program, and by theNIH National Institute of General MedicalSciences. H.J.W. acknowledges Dr A. V. Cherepa-nov for useful discussions and for his advice inusing the Igor Pro software package, and R.Zwier for excellent technical assistance withbuilding the potentiometric equipment.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2006.02.042

The Supplementary data contains the Igor routineto integrate optical spectra.

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Edited by M. Guss

(Received 26 August 2005; received in revised form 13 February 2006; accepted 15 February 2006)Available online 6 March 2006