Heme Ligand Binding Properties and Intradimer Interactionsin the Full-length Sensor Protein Dos from Escherichia coliand Its Isolated Heme Domain*□S

Received for publication, September 21, 2009, and in revised form, October 21, 2009 Published, JBC Papers in Press, October 28, 2009, DOI 10.1074/jbc.M109.066811

Christophe Lechauve‡, Latifa Bouzhir-Sima§¶, Taku Yamashita§¶1, Michael C. Marden‡, Marten H. Vos§¶2,Ursula Liebl§¶, and Laurent Kiger‡3

From ‡INSERM U779, Universites Paris VI et XI, 94276 Le Kremlin-Bicetre, France, the §Laboratory of Optics and Biosciences, CNRS,Ecole Polytechnique, 91128 Palaiseau, France, and ¶INSERM U696, 91228 Palaiseau, France

Dos from Escherichia coli is a bacterial gas sensor proteincomprising a heme-containing gas sensor domain and a phos-phodiesterase catalytic domain. Using a combination of staticlight scattering and gel filtration experiments, we establishedthat, as aremany other sensor proteins, the full-length protein isdimeric. The full-length dimer (association constant <10 nM) ismore stable than the dimeric heme domain (association con-stant�1�M), and the dimer interface presumably includes bothsensor and catalytic domains. Ultrafast spectroscopic studiesshowed little influenceof the catalytic domainonkinetic processesin the direct vicinity of the heme. By contrast, the properties ofligand (CO and O2) binding to the heme in the sensor domain,occurring on amicrosecond to second time scale, were found to beinfluenced by (i) the presence of the catalytic domain, (ii) thedimerization state, and indimers, (iii) the ligation stateof theothersubunit. These results imply allosteric interactions within dimers.Steady-state titrations demonstratedmarked cooperativity in oxy-gen binding to both the full-length protein and the isolated hemedomain, a feature not reported to date for any dimeric sensor pro-tein. Analysis of a variety of time-resolved experiments showedthatMet-95 plays a major role in the intradimer interactions. TheintrinsicbindinganddissociationratesofMet-95 to thehemeweremodulated �10-fold by intradimer and sensor-catalytic domaininteractions. Dimerization effects were also observed for cyanidebinding to the ferric heme domains, suggesting a similar role forMet-95 in ferric proteins.

Dos from Escherichia coli (EcDos)4 is a modular gas sensorprotein in which phosphodiesterase activity is coupled with the

binding and release of external ligands in an associated heme-binding sensor domain (1–3). CyclicAMP (4) and cyclic diGMPact as substrates of this enzyme, the latter with much higheractivity (5).EcDos has been found to display 6–7-fold enhancedcatalytic activity toward cyclic diGMP after binding of gaseousmolecules such as O2, CO, and NO to the heme.

Although the heme-containing PAS sensor domains ofEcDos and the rhizobial sensor protein FixL (6) share a con-served structural fold, the ligand-induced regulation of bothproteins differs considerably. In contrast to EcDos, FixL is anoxygen-specific sensor that couples the status of its sensordomain to the activity of a histidine kinase. This enzymaticactivity is decreased to a great extent upon O2 binding (2). Fur-thermore, whereas in the ferrous deoxy form of FixL the hemeis pentacoordinate, in EcDos the heme iron is coordinated to aproximal histidine (His-77) and a distal methionine (Met-95).Met-95 can be replaced by small gaseous molecules and plays acrucial role in the regulation of the catalytic activity.The three-dimensional structures of the Fe(III), Fe(II), and

Fe(II)-O2 complexes of the isolated heme sensor domain ofEcDosH (EcDosH) have been determined (7, 8). These struc-tures have revealed the importance of several key amino acidsnear the distal heme-binding site involved in changes in theheme environment upon O2 binding. In particular, upon itsreplacement by O2 as a heme ligand, the internal ligand, Met-95, undergoes a major structural rearrangement toward a posi-tion where it points out of the heme pocket. Rearrangementsoccur equally for Arg-97 and Phe-113, which are both involvedin stabilizing the polar Fe–O2 bond by hydrogen bond interac-tions. The crucial roles of these residues in regulating the cata-lytic domain have been studied (9–11). In the ferric form ofcrystallized EcDosH, a water molecule rather than Met-95 isbound in the distal heme site (7).The heme sensor domain EcDosH crystallizes as a dimer (7,

8) and is also dimeric in solution (1). Surprisingly, based on gelfiltration chromatography, full-length EcDos has been charac-terized as a tetramer (12, 13). This contrasts with many otherheme-based sensor enzymes, including FixL or the CO sensorCooA, which have been described as stable dimers (14–17).The reactivity of EcDos to various gaseous signaling ligands

(O2, NO, CO) is determined by their binding and release kinet-ics to the heme domain and by intermediates formed during thekinetic process. For the full-length protein,O2,CO, and cyanidebinding studies on the time scale of seconds have been reported

* This work was supported by INSERM, Delegation Generale pour l’Armement(DGA) Contract 07.34.004, and Universites Paris VI et XI.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 4.

1 Recipient of a European Commission Marie Curie Incoming International Fel-lowship. Present address: Laboratory of Analytical Chemistry, Graduate Schoolof Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.

2 To whom correspondence may be addressed: Laboratoire d’Optique et Bio-sciences, CNRS, Ecole Polytechnique, F-91128 Palaiseau, France. Tel.: 33-1-69-08-50-66; Fax: 33-1-69-08-50-84; E-mail: marten.vos@polytechnique.edu.

3 To whom correspondence may be addressed: INSERM U779, 78 rue du Gen-eral Leclerc, Hopital de Bicetre, Bat. Broca, Niveau 3, 94275 Le Kremlin-Bicetre, France. Tel.: 33-1-49-59-56-64; Fax: 33-1-49-59-56-61; E-mail:Laurent.Kiger@inserm.fr.

4 The abbreviations used are: EcDos, E. coli direct oxygen sensor; EcDosH,heme sensor domain of EcDos; FixLH, heme sensor domain of FixL; PBS,phosphate-buffered saline; MALLS, multi-angle laser light scattering.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 52, pp. 36146 –36159, December 25, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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that indicate an influence of the presence of the enzymaticdomain (10, 18). However, kinetic spectroscopic studies of therelevant processes on the femtosecond to millisecond timescales have been performed to date essentially only on the iso-lated heme domain (1, 11, 17, 19–21).We have now heterologously expressed the full-length pro-

tein EcDos in high quality and quantity suited for detailed staticand time-resolved optical spectroscopic measurements. Here,we present a study of the combined oligomerization and ligandbinding properties of EcDos and its sensor domain EcDosH, aswell as a comparison with the isolated heme domain of FixL(FixLH). We determined the molar masses and hydrodynamicdiameters of the above proteins by size exclusion fast proteinliquid chromatography coupled with multi-angle laser lightscattering (MALLS). We concluded that full-length EcDos is adimeric protein, as are the sensor domains EcDosH and FixLH.The monomer-monomer interfaces for the truncated proteinswere found to be considerably less stable than for the full-lengthprotein. The microscopic binding constants of O2, CO, andMet-95 were measured for EcDos, EcDosH, and two EcDosHmutant proteins with the substitutions M95I and M95H. Wepresent evidence for substantial allosteric interactions betweenthe two proteins constituting the dimer, leading to modulationof ligand binding parameters and in particular to a cooperativemechanism for O2 binding.

MATERIALS AND METHODS

Expression and Purification of EcDos and EcDosH—The genecoding for the full-length protein EcDos (corresponding tocodons 1–799) was amplified by PCR using the primers 5�-atgaag cta acc gat gcg gat-3� (forward) and 5�-tca gat ttt cag cgg taacac-3� (reverse) and subsequently cloned into a pBAD TOPOTA cloning vector (Invitrogen). The construct obtained undercontrol of an arabinose-inducible promoter was verified byDNA sequencing and eventually transformed into E. coliHB101 or BL21DE3 for protein expression in autoinduciblemedium (22) or with 0.2% arabinose induction. 1 mM �-amino-levulinic acid was added to the culture medium. Cultures weregrown for 48 h at 32 °C and 250 rpm agitation. The protein waspurified with an Akta purifier system (GE Healthcare) using a5-ml HisTrap affinity column (GE Healthcare) equilibratedwith PBS, pH 7.5, and was eluted at a concentration of 250 mM

imidazole. The samples obtained were loaded on a desaltingSephadexG-25 column (GEHealthcare) to eliminate imidazoleand subsequently suspended in PBS, pH 7.5, or 50 mM Tris/HCl, pH 7.6.The DNA fragment corresponding to codons 1–142, coding

for the PAS heme domain (EcDosH), was amplified using theprimers 5�-tta ctc cat atg aag cta acc gat gcg gat aa-3� (forward)and 5�-cga gtg gga tcc cta aac ggc aat aat caa ttg tc-3� (reverse)and cloned in pET-3a (Novagen). The final constructs weretransformed into E. coli BL21DE3 for expression in autoinduc-ible medium (Overnight Express Instant TB medium, Nova-gen), and 1 mM �-aminolevulinic acid was added to the culturemedium. Cultures were grown for 24 h at 32 °C and 250 rpmagitation. The EcDosH proteins were purified with an Aktapurifier system on a Hitrap DEAE-Sepharose column (Amer-sham Biosciences). Because of the low pI of EcDosH, the sam-

ples were loaded on the column equilibrated with 50 mM Tris/HCl buffer, pH 8.5, and the protein was eluted at 50 mM NaCl.The concentrated material was loaded on a SuperoseTM 12 HR16/50 (GE Healthcare) column equilibrated with PBS, pH 7.5.Expression and purification of the mutant proteins EcDosHM95I and M95H were carried out as described previously (20).The gene fragment encoding FixLH was amplified and

cloned as described previously (23). The protein was expressedin autoinducible medium (Overnight Express Instant TBmedium), and 1 mM �-aminolevulinic acid was added to theculture medium. Cultures were grown for 24 h at 32 °C and 250rpm agitation. The protein purification protocol was the sameas for EcDos.Pure ferric EcDos, EcDosH, and FixLH were found to have

absorption ratios (A280 nm/AmaxSoret) of 1, 0.36, and 0.24,respectively, very similar to those published previously (1, 13,24). These comparisons, as well as the comparison with pre-dicted spectra based on protein composition (not shown), indi-cate that the heme tomonomer ratio was close to 1 for all of ourpreparations.Gel Filtration—Gel filtration experiments were carried out

using a fast protein liquid chromatograph (Gilson) equippedwith a SuperoseTM 12 HR 10/300 GL column (GE Healthcare)at 25 °C in buffer containing 30 mM PBS, pH 7.5, and 100 mM

NaCl. The elution time was determined at the peak half-height;a void volume of 7.8ml and an internal volume for the gel bed of25 ml were used to calculate the molecular sieve coefficients(Kav). High reproducibility of the loaded sample volumes wasobtained using a Gilson autoinjector device; for an injectionvolume of 70 �l, a dilution factor of 10 after elution was calcu-lated. The absorbance of the eluent was registered at 415 and280 nm.We analyzed the elution profiles for each sample in different

ligation states. The oxygenated forms were studied directlyafter purification to avoid heme oxidation, and 2 mM dithio-threitol was added to ensure full reduction of the heme pro-teins. The oxidized forms were obtained by heme oxidationwith an excess of potassium ferricyanide after deoxygenation ofthe sample, to favor oxidation over O2 binding and to limit thetime of reaction with the oxidant finally removed after loadingthe samples onto a desalting Sephadex G-25 column (GEHealthcare). Completion of cyanide binding to the ferric pro-tein was checked spectrophotometrically.Dynamic Light Scattering—The particle size was measured

with a Zetasizer Nano-ZS (Malvern Instruments), based ondynamic light scattering. Size distribution by volume was usedto interpret the results. Measurements were performed at20 °C in 30 mM PBS, pH 7.5, 0.10 M NaCl, and the diameterwas determined from the average of triplicate measure-ments. Hydrodynamic diameters of the protein were esti-mated relative to those of standard proteins, specificallyrecombinant dehaloperoxidase (15.5 kDa), myoglobin (16.1kDa) (Sigma-Aldrich), recombinant neuroglobin (16.5 kDa),hemoglobin (64.7 kDa), diaspirin-cross-linked hemoglobin(Baxter Healthcare Corp., Deerfield, IL), albumin monomerstandard (67 kDa) (Sigma-Aldrich), and a recombinantoctameric hemoglobin (131.7 kDa).

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Size Exclusion by Fast Protein Liquid Chromatography andMulti-angle Laser Light Scattering—The molar masses in solu-tion were determined by MALLS coupled with size exclusionchromatography (SEC-MALLS). Gel filtration separation reac-tions were carried out using an EttanTM LC liquid chromatog-raphy system (GE Healthcare) equipped with a SuperoseTM 12HR 10/300 GL column (GE Healthcare). Isocratic elution wasperformed at a flow rate of 0.39ml/min using amobile phase of30 mM PBS, pH 7.5, 100 mM NaCl, and 0.03% sodium azide at25 °C. Light scattering analysis was performed usingan EttanTM LC HPLC system with an automatic degasser andthermostatted autosampler, connected in-line to a DAWN�HELEOSTM II 18-angle static light scattering detector; this wasequipped with a quasi-elastic light scattering instrument(QELS;Wyatt Technology, Santa Barbara, CA) and anOptilab�rEX differential refractometer with a Peltier temperature-reg-ulated flow cell maintained at 25 °C (Wyatt Technology, SantaBarbara, CA). Calibration of the light scattering detector wasverified using an albumin monomer standard, recombinantneuroglobin, myoglobin, and diaspirin-cross-linked hemoglo-bin. The molar mass was calculated from the light scatteringdata using a specific refractive index increment (dn/dc) value of0.183 ml/g. The light scattering detector was controlled andanalyzed using ASTRA V software (version 5.3.4.13) (WyattTechnology).Spectra and Ligand Binding/Dissociation Kinetics—Steady-

state spectral measurements were performed with a Varian Cary400 or aHP 8453 diode array spectrophotometer. All ligand bind-ing experiments were performed in 150 mM Tris acetate, 50 mM

NaCl, pH7.5, or inPBS,pH7.5. FivemMdithiothreitolwas addedto all EcDos samples to avoid misfolded proteins and foldingintermediates due to the formation of disulfide bonds.Association rates of cyanide to heme were obtained by mon-

itoring changes of absorption in PBS, pH 7.5, at 25 °C with a HP8453 diode array spectrophotometer. The same apparatus wasused for measuring the CO dissociation from EcDos afterreplacement by either 1 atm of NO or O2.The bimolecular binding kinetics after heme ligand photoly-

sis were measured using a Nd:YAG Big Sky laser CFR-300(Quantel) generating 8 ns/30 mJ pulses at 532 nm. The laserbeam and the detection monochromatic light were directed tothe sample-containing optical cuvette by long length glass opti-cal fibers. We also used a repeat sequence of pulses at 10 Hzfrequency during �10 s to photoincubate the EcDos protein inthe hexacoordinated state by flashing off the CO.Stopped-flow rapid mixing experiments were performed

with SFM-3 Bio-Logic equipment. The methods used to assesshexacoordination and bimolecular CO and O2 rate constantshave been described previously (25). Samples from 1 to 10 �M,as determined on the basis of heme absorption, were measuredin 4-mm optical path length quartz cells, whereas more highlyconcentrated samples (�10 �M) were measured in 1-mm cells.Multicolor femtosecond absorption spectroscopy was per-

formed as described (21) with a 30-fs pump pulse centered at565 nm and a �30-fs white light continuum probe pulse at arepetition rate of 30 Hz and a temperature of 20 °C. The pro-teins were prepared to a sample concentration of 50 to 70 �M.

Equilibrium Oxygen Binding Curves—O2 binding curves atequilibrium were measured using a HEMOX analyzer (TCSScientific Corp.), allowing the simultaneous measurement ofabsorption and oxygen tension by a Clark-type electrode upondeoxygenation with nitrogen. A dual wavelength measurementis monitored at 560 and 576 nm, which closely matches themaximum and minimum of the absorption difference betweenthe O2 and deoxy hexacoordinated spectra. Nevertheless, thecalculation of the fractional saturation can be more complex ifanother spectral component is involved during the deoxygen-ation process, especially for the partially liganded species.Therefore controlmeasurements of thewhole absorption spec-tra in the visible region at different O2 fractional saturationwere performed. Similar O2 binding curve shapes were mea-sured forEcDos andEcDosH, and several isosbestic points werefound. A further complication arises from the rate of auto-ox-idation ofEcDos,which increases at lowpartialO2 pressure. Forthis reason we used an enzymatic systemwith ferredoxin as theterminal electron donor (26), achieving full reduction of theoxidized proteins within 1 min.

RESULTS

Size Exclusion Chromatography and Light Scattering—Thetheoreticalmolarmass ofmonomeric full-lengthEcDos and theheme domain EcDosH is 93,000 (including the His6 tag at amolar mass �2,000 g/mol) and 16,200 g/mol, respectively.EcDos and EcDosH were characterized previously by gel filtra-tion as tetrameric and dimeric, respectively (1, 12). Althoughgel filtration is useful for estimating equilibrium constantsbetween protein subunits, it is considerably less accurate fordetermining the absoluteMr and aggregation state of a protein,unless theMr markers belong to the same protein family as theprotein of interest (see “Discussion”). For this reasonwe used inparallel the static light scattering technique (MALLS), whichallows the measuring of absolute molar masses and sizes ofmoleculeswithout having to rely on the calibration of standardsand assumptions of their conformation.Molar masses determined by MALLS analysis coupled with

size exclusion chromatography yielded aMr of 199,000� 4,000g/mol for the full-length protein EcDos (Fig. 1A) and 32,000 �100 g/mol for both the EcDosH and FixLH heme domains (sup-plemental Fig. 1) for heme concentrations of several �M

(EcDos) and several tens of �M (EcDosH and FixLH). No evi-dence of a tetrameric structure was found for any sample, evenat high concentrations (�40 �M; instead, our data indicateddimeric forms for all three proteins. The heme domainsEcDosH and FixLH show a marked dependence on the proteinsize as measured by gel filtration in the heme concentrationrange of 40 to 0.05 �M, indicating a change in their oligomeri-zation state (Fig. 2). The elution profile for EcDos is concentra-tion-independent in the range of 1 to 0.01 �M (Fig. 2A).We estimated the hydrodynamic radius (Rh) using two

dynamic light scattering instruments (Wyatt Technology andMalvern Instruments). The Rh values for monomeric EcDosH(2.3 nm), dimeric EcDosH (2.8 nm), and full-length EcDos (6.5nm) were plotted against their Mr along with several globularproteins (Fig. 1B). Different from FixLH, the values for EcDosHand EcDos do not appear to correlate well with the linear rela-

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tion found betweenRh andMr for the other proteins, suggestinga less compact and globular-like geometry of EcDosH comparedwith FixLH and the globins used for comparison. The interpreta-tion of gel filtration experiments, assuming a direct relationbetween hydrodynamic volumes and Mr, explains the previousassessmentof a tetrameric structure forEcDos (12, 13). ForEcDos,the Rh distribution (at a few �M) was identical in the ferric andferroushexacoordinated form, aswell as in theCOferrous form. Itis therefore reasonable to assume that EcDos was fully dimeric inour functional studies whatever the ligation state of the heme

domain. Because three-dimensionalstructures are only available for theheme domain of EcDos, we cannotassess to date whether the differencein the stability of the dimeric struc-ture between EcDosH and EcDosstems from a change at the hemedomain interface or rather from theadditional interface between the twoenzymatic domains.Fig. 2, B and C, shows the parti-

tion coefficients measured by gelfiltration chromatography forEcDosH and FixLH in the ferric(Fe(III)), ferric cyanide (Fe(III)-CN),and ferrous oxy forms. The equilib-rium binding constants for associa-tion of monomers into dimers were1 and 0.4 �M for EcDosH and 0.8and 1.4 �M for FixLH in theiroxidized and oxygenated forms,respectively. This difference repre-sents a stabilization of the EcDosHheme domain interface of 0.5 kcal/mol upon oxygen binding (subunitassociation free energy: �8.7 kcal/mol), whereas for cyanide binding,the energy change is very smallcompared with the hexacoordi-nated oxidized form (Fig. 2B). Thisenergy change obviously resultsfrom a structural change at the sub-unit interface. FixLH presents aweak destabilization of the hemedomain interface of 0.35 kcal/molfor oxygen binding (�8 kcal/mol)with no difference in free energy atthe interface upon cyanide bindingcompared with the pentacoordi-nated oxidized form.Cyanide Binding Kinetics—To

further investigate the nature of thedistal ligand in the ferric state inEcDos proteins in solution (a watermolecule in the EcDosH crystalstructures (7, 8)), we compared thecyanide binding kinetics of EcDos,EcDosH, the M95I mutant, and

myoglobin. The binding kinetics of cyanide were pseudo-firstorder. We did not observe any biphasic pattern indicative of aheme in equilibrium between significant amounts of pentaco-ordinated (or hexacoordinated to a low affinity ferric ligandeither a water molecule or a hydroxide) and a strong hexacoor-dinated state with the methionine. The bimolecular cyanidebinding rates were, respectively, 117 M�1�s�1 for myoglobin;113 M�1�s�1 for M95I EcDosH; 30.5 M�1�s�1 for EcDosH at 0.3�M and 9 M�1�s�1 for EcDosH at 17 �M; and 0.9 M�1�s�1 forfull-length EcDos (supplemental Fig. 2). Overall, these rates are

FIGURE 1. A, molar mass measured by multi-angle light scattering after gel filtration for EcDos (5 �M) usingSEC-MALL. B, correlation between hydrodynamic diameters (measured by dynamic light scattering, MalvernInstrument and Wyatt Technology) and the molar mass of six globular proteins (E). FixLH hydrodynamicdiameters (monomeric, 0.5 �M; dimeric, 25 �M) (F) fit well with the hydrodynamic diameters of globularproteins, whereas monomeric (0.5 �M) and dimeric (30 �M) EcDosH PAS (Œ) and full-length EcDos (f) hydro-dynamic diameters deviate from the correlation line.

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in the same range as thosemeasuredpreviously (19, 27), although quan-titative differences were observed(see “Discussion”).We note that therate for the M95I EcDosH mutantprotein is very similar to that formyoglobin, consistent with a similarligation of the ferric heme. Thebinding rate forEcDosH ismarkedlylower. Our results show that forEcDosH the association ratesdepend on the protein quaternarystate (monomer/dimer). Under thehypothesis that in ferric EcDosH insolution the heme would be coordi-natingMet-95, this differencemay beexplained by the difference in themicroscopic association anddissocia-tion rates for the methionine residuebetween the EcDosH monomer anddimer (see below), with the EcDosHdimerexhibiting ligandbindingprop-erties closer to those of the full-lengthproteinEcDos (see also “Discussion”).Ultrafast External and Internal

Ligand Rebinding Kinetics—Thedynamics of internal and externalligand binding in the heme domainwere investigated using femtosec-ond spectroscopy under conditionsin which all investigated proteinswere predominantly dimeric. Fig. 3compares the kinetics of rebindingof the internal ligand, Met-95, andCO after photolysis from the hemein EcDosH and in the full-lengthprotein EcDos. The Met-95 rebind-ing kinetics are biexponential withtime constants of �7 and �35 ps(20), presumably reflecting two con-figurations of Met-95 that do notstrongly interchange on the timescale of the experiment (21). In thefull-length protein the overall re-binding occurs moderately, but sig-nificantly, faster than in the isolatedheme domain (Fig. 3A). In particu-lar, the relative amplitude of thefaster component is higher. Thiseffect is qualitatively similar to theeffect of glycerol on the hemedomain (21) and suggests that thepresence of the enzymatic domaininfluences the relative population ofthe two Met-95 configurations inthe heme domain.In contrast, the partial geminate

rebinding of CO to the heme occur-

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ring on the picosecond-to-nanosecond time scale is very similar inEcDosH and EcDos (Fig. 3B). These kinetics reflect competitionbetween low energy barrier rebinding to the heme and thermallyactivated escape from the heme pocket (21), suggesting that theinitial escape route for CO is not influenced by the enzymaticdomain.

Microsecond to Second CORebindingKinetics for EcDosH—Fig.4A shows that the shape of the tran-sient absorption spectrum mea-sured at 0.1 �s for EcDos andEcDosH is close to that measured at2 orders of magnitude faster time.After CO migration out of the pro-tein, Met-95 can rebind on the 100�s time scale, in competition withbimolecular CO rebinding (20). Theshape of the transient spectrumchanges and is well simulated by thedifference of the steady-state spec-tra: deoxy hexacoordinated His-heme-Met minus His-heme-CO(Fig. 4A). Subsequent replacementof Met-95 by CO from solutiontakes place in milliseconds to sec-onds (20). To investigate theinfluence of dimerization on theintrinsic rate constants for Met-95and CO binding and dissociation inEcDosH, we monitored Met-95 andCO binding kinetics after flash pho-tolysis as a function of the CO con-centration for two different hemeconcentrations. At 35�M EcDosH isalmost exclusively dimeric, whereasat 1 �M it is significantly displacedtoward the monomeric form (seeabove).Any proteins with hemes coordi-

nating not CO but Met-95 prior tothe flash will not contribute to thesignal, because after flash photolysisMet-95 rebinds (Fig. 3A) on a sub-nanosecond time scale. Equilibra-tion with 0.01 atm of CO leads tocomplete CO saturation of the pro-tein. The rebinding occurs in twomain phases reflecting the abovedescribed binding and replacementprocesses under all conditions.However, the dependence of thekinetics on both protein and COconcentration (Fig. 4B) is complex.For the fully dimeric CO-saturated

samples, the kinetics of each phase can be satisfactorilydescribed by a single exponential. For the mixed dimeric/mo-nomeric samples, we observed two rates in the binding phaseand replacement phase each, at all CO concentrations. Thus,the microscopic reaction rates are different in the monomericand dimeric heme domains. Analysis of the CO dependence on

FIGURE 2. A, normalized gel filtration elution profiles of EcDos (1 �M (– – –) and 0.01 �M (����)) and the heme domain, EcDosH, in a concentration range between15 and 0.05 �M. B and C, protein size dependence on concentration, as measured by gel filtration. Molecular sieve coefficients, Kav, are plotted versus proteinconcentration for EcDosH (B) and FixLH (C). Three different forms of each protein were plotted: oxy-ferrous (Œ), cyanide-ferric (f), and ferric (F).

FIGURE 3. Transient absorption experiments showing geminate rebinding of Met-95 in fully reduced (A)and of CO (decay, 64%; time constant, 1.6 ns) in reduced CO-bound (B) EcDos (F) and EcDosH (E). Thelines in A indicate fits with biexponential decays with time constants of 6 and 30 ps (58 and 42%) and 7 and 35ps (53 and 47%) for EcDos and EcDosH, respectively.

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the binding reaction allows us to extract the konMet and konCOvalues, which then permits the estimation of the koffMet valuewith analytical and numerical approaches as described previ-ously for other hexacoordinated heme proteins (25). This anal-ysis indicates that konCO is 5.106 M�1�s�1 for both monomersand dimers, a value very similar to that determined previously(20). On the other hand, konMet and koffMet are both 1 order ofmagnitude higher for monomers (30,000 and 400 s�1) than forCO-saturated (see below) dimers (2000 and 50 s�1) (Table 1).

Thus, whereas the affinities formethionine are close for monomersand dimers, the monomer bindingrates of Met-95 are 10 times faster,indicating a more flexible hemepocket.Interestingly, the contribution of

a faster reactive conformation doesnot increase only with a decrease inthe protein concentration but alsoat a low CO saturation level for highprotein concentration (data notshown). This indicates that themonoliganded species (one CO perEcDosH dimer) behaves differentlythan the diliganded species (konMet30,000 s�1 versus 2,000 s�1; koffMetis less affected) and displays Met-95binding similar to the monomer ofEcDosH. This implies the presenceof allosteric interactions betweenthe two hemes of the dimer. Forinstance at 10% CO-heme satura-tion, the faster rebinding rate repre-sents only 30% of the binding phase.Given that the dominant ligandedspecies is the singly liganded form(90% for a binomial distribution,less for a cooperative system), thisresult reflects the allosteric equilib-rium of the singly liganded dimerwith at least one-third in the rapidstate for Met rebinding.Microsecond to Second CO

Rebinding Kinetics for EcDos—Theabsorption spectra of Fe(III), Fe(II),and Fe(II)-CO (0.01, 0.1, and 1 atm)and of Fe(II)-O2 complexes ofEcDos are shown in Fig. 5A. Equili-bration with 0.01 and 0.1 atm of COleads to incomplete CO saturationof the protein with, respectively, a25 and 5%presence of the hexacoor-dinated Met-95-bound form.The characteristics of the CO

rebinding kinetics for the full-length protein EcDos (Fig. 5B)resemble those measured forEcDosH at a high rather than a low

protein concentration. This observation is consistent with thefinding that EcDos is fully dimeric (see above). At 1 atm of CO(full CO saturation), the kinetics of both phases are close tosingle exponential. At high CO concentration, the bindingphase occurs predominantly at a [CO]-dependent rate (�6000s�1 at 1 atm of CO; Fig. 6A). Despite the full CO saturation, thebinding of a few percentages of the heme occurs much faster atan almost [CO]-independent rate of 30,000 s�1 (see below).The Met-95 replacement phase is 1 order of magnitude slower

FIGURE 4. A, transient absorption spectra after flash photolysis of the heme-CO complex of EcDos (‚) andEcDosH (�) at 0. 1 �s and ms time scales. – – –, transient spectrum at 4 ns; —, difference between steady-statespectra: deoxy hexacoordinated His-Fe2�-Met minus His-Fe2�-CO. B, flash photolysis kinetics for EcDosH at 436nm and 25 °C. Recombination kinetics at different CO concentrations, from top to bottom: 0.01, 0.1, and 1 atmof CO. After flash photolysis of CO, the first phase represents a competitive binding between CO and Met to theheme. The second phase is a slow replacement reaction of Met by CO to return to the preflash steady state.Heme concentration dependence on CO kinetics is observed, due to the presence of two protein structures inequilibrium: monomeric and dimeric with different Met on- and off-rates.

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than inEcDosH (Fig. 4B) and limited by the koffMet (�2 s�1). Atlower CO concentrations the kinetics become increasinglymulti-exponential.We attribute this to the fact that the samplesare not fully CO-saturated and contain mixtures of diligandedandmonoliganded dimers with different kinetic parameters, inparticular konMet (any dimers not binding CO do not contrib-ute to the signal at this time scale). Analysis of the ensemble ofdata indicates that for both configurations the konCO is 5.106M�1�s�1 (close to the value found forEcDosH) and the konMet is2,000 s�1 (di-CO dimers) (Fig. 6A and Table 1) and 30,000 s�1

(mono-CO dimers) as in EcDosH. The fact that the allostericeffect of CO binding in the two proteins of the dimer is verysimilar for EcDosH and full-length EcDos indicates that thedimer interface between the heme domains is similar for bothproteins. Our results clearly indicate a conformational differ-ence at the dimer interface between di- andmono-CO-contain-ing dimers. Such a difference can be expected also for unligand-ed ferrous dimers. Therefore, a change in conformation and inthe associated konMet is expected to occur after CO dissocia-tion. The finding that strong differences in kinetics between di-and mono-CO-containing dimers are observed at the submilli-second time range indicates that these changes occur in a rangelonger than milliseconds. Indeed, in our previous work on CObinding to EcDosH (20), we had already noted a difference inthe replacement phase between flash photolysis and stopped-flow mixing (starting from fully unliganded protein) that weattributed to a slow conformational change. Such a differenceis also observed in the full-length protein (Fig. 6B). To fur-ther investigate the time scale of the conformationalchanges, we performed photoincubation experiments inwhich the sample was brought to an unliganded state(hexacoordinate Met-95 bound) by a series of laser flashes at10 Hz, and the replacement reaction was subsequently mon-itored. In single-flash experiments this replacement takesplace in �0.5 s at 1 atm of CO to �10 s at 0.01 atm of CO(Figs. 5B and 6B), limiting the time of the unliganded pro-teins for possible conformational changes. Note that at 0.01atm of CO, after a single-flash photodissociation, the Metreplacement reaction by CO is biphasic, because in the unli-ganded form the transition between the fast and the slowreactive conformation occurs on the seconds time scale

(only 30% of the fast component was measured). Note thatidentical results were obtained in stopped-flow and photo-incubation experiments (Fig. 6B); the rates were unchangedfor 1 atm of CO and decreased 6-fold for 0.01 atm of COcompared with flash photolysis experiments. The conforma-tional change at a low CO concentration is most significant,as at 1 atm of CO the rate is limited by the koffMet. Weconclude that the conformational change takes place on thetime scale of a few seconds or faster. In fact the curves couldbe fitted well with the kon and koff values for methioninekeeping the same CO kon and koff values measured for theliganded EcDos form. konCO was measured by flash photol-ysis after subtracting the contribution of konMet determinedin Fig. 6A, whereas koffCO was measured by replacing it witha large excess of NO or O2 (data not shown). For the slowreactive conformation with a konMet value of 30000 s�1,measured for the partially liganded species, the replacementrate data set was simulated with a koffMet of 9 s�1.In EcDosH, in similar photoinduction experiments, or after

mixing with CO by stopped-flow, the binding kinetics are alsoslower than those measured after single-pulse flash photolysis(supplemental Fig. 3) (20). This implies that EcDosH also rapidlyreaches another conformational state after external ligandremoval.The microscopic rate values are summarized in Table 1. On

the basis of the kinetic experiments, EcDos shows evidence ofdifferent reactive states for methionine depending on the liga-tion states with CO. At least two different states are involvedduring the overall ligand binding reaction with CO for whichmethionine affinity differs by a factor of 3 to 4.Microsecond to Second O2 Rebinding Kinetics for EcDos and

EcDosH—TheO2 binding and dissociation kinetics were inves-tigated using CO-liganded samples in the presence of O2. Byanalogy with the above described photoincubation experi-ments, we chose experimental conditions that allowO2 to com-petewithCO for heme rebinding after flash photolysis. Becausethe yield of O2 escape to the solvent after heme-O2 dissociationis very low, the bimolecular reactionwithO2 can best be studiedupon CO photodissociation in the presence of O2; O2 will thenbind, and eventually be replaced by CO (supplemental Fig. 4).Themicroscopic binding constants kon and koff for the external

TABLE 1O2 and CO binding parameters for full-length EcDos and the EcDosH sensor domainThe experimental conditions are: 150 mM Tris acetate, 50 mM NaCl, pH 7.5, at 25 °C (20 °C for BjFixL).

Protein konCO koffCO KCOa konO2 koffO2 KO2a konMet koffMet KMet

/M/s /s mmHg /M/s /s mmHg /s /sEcDos 4 � 106 0.007 1.3/3.0b 2.0 � 107 1.1 30/(11/69)c 2,000 2 1,000

30,000 9d 3,300EcDosH monomer 5 � 106 2.0 � 107 30,000 400 75EcDosH dimer 5 � 106 2.0 � 107 1.5 1.6/(1.6/11)c 2,000 50 40

30,000 70d 430dEcDosHM95I 3.4 � 106 2.0 � 107 1.2 0.03EcDosHM95H 8 � 106 3.0 � 107 2.1 0.2 150(His) 40(His) 4(His)BjFixLH 1.6 � 104 1.9 � 105 6 18

a Because of the competition for heme binding between CO or O2 and Met, the overall ligand affinities differ from the intrinsic affinities (kon/koff) and are calculated from thefollowing formula: Kligand (kon ligand/koff ligand)/(1 � KMet). Solubilities are 1.82 10�6 �M/mmHg and 1.36 10�6 �M/mmHg for O2 and CO, respectively.

b This equilibrium constant corresponds to the CO partial pressure of the binding sites at half-saturation.c In parentheses are shownO2 affinities measured at equilibrium for the two binding steps. Note that the oxygen binding enthalpy was measured at equilibrium equal to �9.0 �0.5 kcal/mol.

d Estimated from CO binding kinetics by a combination of stopped-flowmeasurements to probe the unliganded state (see “Results”) and flash photolysis measurements for thebinding rates of CO andMet. For this latter value, we used the fastMet binding rate measured for the partially liganded species (see “Results”). The other values present in thistable were measured by flash photolysis from the fully CO-liganded species.

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ligand O2 (and CO) are almost identical for EcDosH and EcDos(Table 1). Thus the intrinsic O2 binding affinities are also sim-ilar. The measured overall binding affinities (P50) depend bothon the competitionwith theMet-95 residue and on the intrinsicO2 affinity and are therefore different for EcDosH and EcDos(Table 1). The intrinsic O2 affinities for EcDosH and EcDos are�3 orders ofmagnitude higher than FixLH and full-length FixL

(24). Although it is obvious that thegas-sensing PAS domains in FixLand EcDos tune their (low) O2 affin-ities by different mechanisms,namely, large steric hindrance forligand binding in FixL and competi-tion with a constitutive internal res-idue for heme binding in EcDos,they nevertheless exhibit very simi-lar overall affinities for oxygen.Microsecond to Second Ligand

Rebinding Kinetics for EcDosHMutants M95I and M95H—Substi-tution of Met-95 by isoleucine givesrise to a pentacoordinated heme inthe deoxy state (19, 21). This allowsthe rate of ligand binding to heme tobe measured directly without com-petition with an internal ligand.Indeed, in contrast to wild typeEcDosH (Fig. 4B), in the M95Imutant protein, the kinetics ofbimolecular CO rebinding afterphotolysis are monophasic. Therates we determined for CO and O2bimolecular binding and dissocia-tion (in the same order as thosedetermined by Gonzalez et al. (19))are very similar to those of wild typeEcDosH (Table 1), as is CO gemi-nate rebinding (21). Thus the pres-ence of Met-95 does not influencethe intrinsic ligand affinities. In par-ticular, the high intrinsic oxygenaffinity is confirmed by these directmeasurements, indicating that theheme pocket is designed for a stableO2 binding. It has been shown thatthe distal arginine 97 acts as a keydeterminant in the heme pocketbinding by forming a hydrogenbound with the bound O2molecule.Mutation of this residue leads to alarge increase in the O2 dissociationrate and thus to auto-oxidation (28)similar to that observed for the anal-ogous arginine 220 in the PASdomain of FixL (29).In theEcDosHmutantM95H, the

histidine residue is able to form areversible bond with the heme (17,

21) but ismuch less flexible than the nativemethionine residue,aswitnessed by the differences in ultrafast binding kinetics (21).To investigate the influence of this property on ligand replace-ment reactions, we determined the bimolecular binding ratesfor CO and O2, as well as the His-95 binding and dissociationrates, in amanner similar towild typeEcDosH (Table 1). Intrin-sic CO and O2 binding was found to be similar to the wild type

FIGURE 5. A, absorbance spectra of EcDos in 150 mM acetate buffer, 50 mM NaCl, pH 7.5. – – –, ferric hexacoor-dinated form; (����, deoxy ferrous hexacoordinated form; ��—��, CO form at 0.01, 0.1, and 1 atm and ferrous oxyform. B, flash photolysis kinetics measurement for EcDos at 438 nm and 25 °C. Recombination kinetics atdifferent CO concentrations is shown (from top to bottom: 0.01, 0.1, and 1 atm of CO). After flash photolysis ofCO, the first phase represents competitive binding between CO and Met to the heme sites. The second phaseis a slow replacement reaction of Met by CO to return to the preflash steady state.

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sensor domain. By contrast, the histidine binding affinity forthe ferrous heme is lower than for methionine because of alarge decrease of the residue association rate from themicro-second to the millisecond range. This finding indicates that thegreater flexibility ofMet-95 overHis-95 allowsmore rapid switch-

ing in the sensor domain. It should benoted that in globin proteins the fast-est His binding rate is also about 1ms(25).Oxygen Binding Curves at Equi-

librium—Fig. 7 shows the oxy-gen binding curve for EcDos.Remarkably, it does not follow asimple nHill 1 binding curve butrequires a two-binding sitemodel. Agood fit was obtained with twointrinsic oxygen binding affinitiesdiffering by a factor of about 6 (11and 69 torr). This compares reason-ably well with the twoMet affinitiesfrom the CO rebinding kinetics,which differ by a factor of 3 to 4.Consequently, the O2 binding of afirst ligand to EcDos gives rise to acooperative binding for the secondligand. The cooperativity index(nHill) reaches a maximum value of1.5 at half-saturation (comparedwith the maximum allowed value of2). This also implies that at equilib-rium the monoliganded species isalways weakly populated withrespect to the sum of the diligandedand unliganded hexacoordinatedspecies. The same behavior wasobserved for EcDosH dimers (Fig.7). Two intrinsic oxygen bindingaffinities (1.6 and 11 torr) wereobserved that were lower than thosefor the full-length protein, in agree-ment with the kinetic data. IndeedEcDosH and the full-length proteinEcDos exhibit the same intrinsicbinding constants except for themethionine dissociation rate, whichis 1 order of magnitude faster forEcDosH, leading to a decrease in theinternal ligand affinity and a lessefficient competition with O2 forheme binding.A summary of the data obtained

on bimolecular binding is presentedin Table 1 and in Fig. 8. Flash pho-tolysis probes the ligand affinity ofthe liganded state, whereas the oxy-gen affinity measured at equilib-rium reveals cooperative behavior.Although ligand binding data have

been measured for EcDosH and EcDos (18, 19), a direct com-parison is difficult because these studies did not take intoaccount the complexities we have revealed here: (i) the func-tional differences between EcDosH monomers and dimers;(ii) the slow protein relaxation upon ligand release, which is

FIGURE 6. A, fast phase rate for the CO binding kinetics versus [CO] for EcDos. As this phase represents thesum of konMet � konCO at low [CO], one expects the curve to reach a plateau if the Met associationbecomes the rate-limiting step (konMet �� konCO). By contrast, at high [CO] konMet becomes negligible;the asymptote of the hyperbola gives the bimolecular rate. Note that the faster rate for the Met (30,000s�1), which increases in amplitude at low [CO] as much as the unbound fraction increased at equilibrium,is not shown. Indeed, in this case konMet is much higher than konCO in the range of [CO] investigated, andno [CO] dependence is measurable. B, rate of the slow phase corresponding to methionine replacementby CO by flash photolysis (‚, upper curve). The lower curve shows the rates of methionine replacement byCO measured by stopped-flow (deoxygenated samples are mixed with a CO-equilibrated buffer) (F) andby photoincubation of the deoxy form with repeat flash cycle (10-Hz laser frequency during severalseconds) (E). The difference between these curves suggests the presence of two conformations for Metbinding to heme with regard to the initial ligation state of the deoxy heme versus liganded heme CO. Inset,replacement of methionine by CO for EcDos after photoinduction (10 s, 10-Hz laser frequency) at differentCO concentrations.

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the basis of ligand binding cooperativity; and finally, (iii) thefunctional differences between the mono- and diligandedspecies, both of which have properties that support ligandbinding cooperativity.

DISCUSSION

Different from the heme domain, we expressed the full-lengthEcDos sensor protein froma construct that puts the geneunder the control of a bacterial pBAD promoter that can beregulated by arabinose. The thusly obtained stable EcDos inhigh quality and quantity in solution allowed an extensivecomparison with the isolated heme domain of both its staticproperties and its fast kinetic processes. Most importantly,we show that the full-length protein is dimeric in solution, asis the heme domain at high concentrations, and that alloste-ric interactions exist between the two units, allowing coop-erative binding of sensed ligands. These points are discussedin more detail below.Quaternary Characterization of EcDos, EcDosH, and FixLH—

Many heme sensor proteins such as FixLH, CooA, and solubleguanylate cyclases are known to be homo- or heterodimeric(14–17). The heme-containing EcDosH sensor domain was

characterized initially as a dimerwith amolar mass of native EcDosHof�36 kDa based upon gel filtrationassays (1).We have nowdeterminedthat the association constants forvarious forms of EcDosH are�1�M

as is also the case for FixLH (Fig. 2).Characterization of the quater-

nary structure of the full-lengthprotein EcDos and the heme sensorPAS domainsEcDosH and FixLHbya combination of analytical gelchromatography and static anddynamic light scattering allowed usto show that the full-length proteinEcDos is a dimeric protein in solu-tion (Fig. 2A). This result contrastswith the previous assessment bySasakura et al. (12) of a tetramericstructure based on gel filtrationchromatography, which assumes alinear correlation between molarmass and elution volumes of proteinmarkers. No correlation betweenthe hydrodynamic radius and themolar mass of EcDos and EcDosHcould be determined using differentproteins from the globin family ascomparison (Fig. 2B). This might bebecause of the less compact overallstructure of the EcDos proteins,which would explain the poor accu-racy in estimating the Mr of theseproteins based on gel filtrationchromatography. Our static lightscattering experiments unambigu-

ously characterized the complex in solution as dimeric.The association constant for full-length EcDos is less than 10

nM, implying that the dimer-dimer interface ismore stable thanin isolated PAS domains (�1 �M). The similarity of theobserved allosteric ligand binding interactions between iso-lated heme domains and full-length EcDos (see below) suggeststhat the dimer interface between the heme domains in bothcomplexes is similar. A plausible explanation for the observa-tion of amore stable full-length dimer is an additional intersub-unit interface arising from additional protein parts, notably thecatalytic domain (no three-dimensional structure is availablefor the full-length protein EcDos). In the available crystal struc-tures of dimeric EcDosH (7, 8), the C-terminal �-strands areoriented in the same direction, so that the two catalyticdomains are likely to share an interaction surface.In EcDosH and FixLH the monomer-dimer equilibrium is

also influenced by the nature of the ligand in the heme pocket.Binding of O2 represents a stabilization of the heme domaininterface in EcDosH, whereas it destabilizes the FixLH hemedomain interface (Fig. 2, B and C). There is a weaker transitionupon cyanide binding to the hexacoordinated form of EcDosHand no change is observed for FixLH. The modulation of the

FIGURE 7. Oxygen binding curve for EcDos and EcDosH at [heme] > 10 �M in PBS buffer (with 2 mM

dithiothreitol for EcDos) and the enzymatic reducing system from Hayashi et al. (26). The data are simu-lated with a two-binding site model (dark line). The dashed straight lines with a slope of unity indicate the twooxygen affinities at the intercept with the x axis.

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monomer-monomer interface due to the ligation state canprobably be explained by the rearrangement of key amino acidsthat occurs in the heme pocket of EcDosH and FixLH (8,29–31). Cusanovich and Meyer (32) suggested that a signalingevent initiated within a PAS domain is transmitted from theligand-binding site to the dimer interface via distortion of thecentral �-sheet to the interacting N and C termini (involved inmonomer-monomer contacts) and their associated hydropho-bic residues. For FixLH it has been suggested that oxygen bind-ing to the hememay result in steric hindrance, whichmay causedistortion of the central �-sheet and alter its interaction withtheN- andC-terminal helices (32), although recently Ayers andMoffat (33) showedminimal crystal lattice effects on the integ-rity of the CO-bound FixLH structure. Binding of differentligands to the heme causesmodulation of themonomer-mono-mer interface of the PAS sensor domain, leading to stabilizationor destabilization. As the interaction of PAS domains is knownto influence the activity of the catalytic domain (13), this findingmay play a role in signaling to the catalytic domain in the stabledimeric full-length proteins.Cyanide Binding and Heme Coordination State in the

Absence of External Ligand—Our data show that dimerizationeffects on ligand binding properties occur not only in ferrousproteins, but also in ferric EcDosH, for cyanide binding (sup-plemental Fig. 2). This finding may explain moderate differ-ences in cyanide binding constants reported by Gonzalez et al.(19) andWatanabe et al. (27), where themonomer-dimer equi-librium was not taken into account.In view of the above discussed role of Met-95 in the dimer-

ization effects of ligand binding in the ferrous species, such arole might also be inferred for the ferric species. In the ferrousspecies, the allosteric effect is most likely brought about by the

large rearrangement imposed by the replacement of Met-95 byan external ligand. However, in crystals, the heme in ferricEcDosH ligates a water molecule rather than Met-95 (7), and alarge rearrangement ofMet-95 upon binding external ligands isnot expected to occur. Spectroscopic investigations indicatethat in solution Met-95 may possibly bind to the ferric heme(17, 19). The literature concerning the steady-state spectro-scopic resolution of this issue, dating from before the x-raystructure determinations, is conflicting (17, 19, 34, 35). Theincrease in the rate of cyanide binding to ferric EcDosH uponreplacement of Met-95 by isoleucine that we measured, alsoobserved upon replacement of Met-95 by alanine and leucine(27), is consistent with the suggestion that Met-95 binds to theheme and is more difficult to replace by cyanide than a watermolecule or a hydroxide, ligating to the heme in the mutantproteins. Altogether, our observations have the potential toreopen the question of the heme ligation state of ferric EcDosproteins.Conformational Changes for EcDos upon LigandDissociation

and Association—Picosecond and early nanosecond kineticevents initiated by ligand dissociation from the heme reflectprocesses in the heme environment prior to ligand exchangewith the protein environment (36). Our ultrafast kinetics didnot show strong differences between the internal dynamics ofEcDosH and EcDos protein (Fig. 3), indicating that the enzy-matic domain does not play a significant role in the earlymolec-ular signaling events which occur after dissociation of methio-nine or an external ligand from the heme. For both, EcDosHand EcDos, we observed two decay constants (7 and 35 ps) formethionine rebinding, arising from the possible presence oftwo configurations of this residue in the heme domain, one ofwhich has been proposed to be active in signal transmission

FIGURE 8. Schemes for CO and Met-95 binding with monomeric EcDosH (A), dimeric EcDosH (B), and full-length EcDos (C) containing the heme domainin tandem with the phosphodiesterase domain (PDE). The rate constants were deduced from CO flash photolysis experiments from the microsecond-to-second time scales. The different Met-95 positions depict two allosteric states. For simplicity, the geminate CO rebinding phases and the picosecond Met-95binding phases are not included in the scheme nor are the intradimer interactions between mixed CO-liganded and nonliganded heme domains.

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(21). The finding that the relative population of these steady-state configurations depends (moderately) on the presence ofthe catalytic domain is fully consistent with this proposal, as itsuggests a mechanistic link between the Met-95 configurationand the catalytic domain.When the heme binds an external ligand, Met-95 is swapped

out of the heme pocket (7, 8). Dissociation of CO then leads tobinding of Met-95 (konMet rate) involving an important reor-ganization event and hence taking place on amuch slower timescale (microseconds) than after dissociation ofMet-95 (20). Forboth dimeric proteins, two Met binding rates also were mea-sured after CO photodissociation (33 and 500 �s), althoughthese were not necessarily related to the two phases observed inthe ultrafastMet-95 dissociation experiments.However, for theEcDosHmonomer only the faster rebinding rate was observed,leading us to propose that the dimeric structure is necessary forat least one conformation. Interestingly, we found that themono-CO-liganded species and the bi-CO-liganded speciesbehave differently with regard to methionine rebinding; theliganded subunit of the asymmetric (CO unliganded) dimer inboth EcDosH and EcDos rebinds methionine with a rate closeto thatmeasured for the EcDosHmonomer.We also found thatthe methionine dissociation rate was affected by the dimeriza-tion and the ligation state. Probing of the hexacoordinated spe-cies reactivity for ligand rebinding after prolonged CO dissoci-ation from EcDosH and EcDos (by photoincubation or simplyafter mixing the deoxy species with a CO buffered solution bystopped-flow) showed a decrease in methionine replacementby the external ligand. If we assume that the binding rates forthe external ligand do not change, this involves a concomitantchange for both the association and dissociation rates of themethionine residue. Indeed, this assumption is supported bythe finding that the other binding parameters we measured forCO and O2 for the EcDosH monomer and dimer, as well as forfull-length EcDos after photodissociation of the fully ligandedprotein-CO complexes, were identical. Altogether our experi-ments thus strongly indicate that the intrinsic parameters gov-erning the binding of external ligands to the heme do notstrongly “sense” the environment of the heme domain but that,by contrast, themethionine dynamic parameters are influencedby (i) the presence of themonomer-monomer interface, (ii) theligation state of the dimer (one or two ligands), and (iii) thepresence of the enzymatic domain because the methionineaffinity increases for EcDos by 1 order of magnitude comparedwith EcDosH. This marked role of methionine in allostericinteractions is in line with the above discussed link with thecatalytic domain.The binding properties mediated by the dynamics of methi-

onine indicate a heme-heme interaction mechanism uponligand binding or release. Indeed theO2 bindingmeasurementsat equilibrium demonstrate unambiguously the presence ofpositive cooperativity. The second oxygenmoleculewill bind toEcDos with a 6-fold higher intrinsic affinity than the first one,and the fraction of the asymmetric monoliganded species willbe much lower than that with two ligands or no ligand at all,whatever the oxygen fractional saturation. On a molecularlevel, this indicates that the replacement of Met-95 by O2 andits swapping out of the heme pocket in one unit lead to a mod-

ification at the dimer interface, presumably leading todecreased Met-95 affinity for heme (and hence increased netO2 affinity) in the other subunit. In view of the predominantrole of Met-95 in the allosteric effects, we suggest that bindingof other ligands (CO andNO) is also cooperative, although theyare more difficult to measure experimentally.There is no doubt that the heme ligand binding properties

will greatly influence enzymatic catalysis in vivo, because adecrease of c-diGMPphosphodiesterase activities upon oxygenrelease has been demonstrated (9). A cooperative mechanismwill obviously enhance the sensitivity to perceive and react tochanges of the ambient tension ofO2 (and other sensed ligands)in the natural environment of E. coli (). To our knowledge, ourassessment of cooperative O2 binding in EcDos constitutes thefirst direct evidence for cooperativity in a heme-based sensorprotein.More generally, only a few examples of cooperativity ina dimeric heme protein have been reported thus far, for a globinhomodimer from the mollusc Scapharca inaequivalis (37) anda truncated globin homodimer from Mycobacterium tubercu-losis (38).

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Ligand Binding in Dimeric EcDos

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FIGURE LEGENDS

Sup. Mat. 1. MW measured by multi-angle light-scattering after gel filtration on a Superose 12 HR 10/300 GL column for the heme domains EcDosH (A) and FixLH (B) using SEC-MALL.

Sup. Mat. 2. Concentration dependence of the first order rate constants for cyanide binding to full length EcDos (0.9 M-1.s-1), EcDosH (0.3 µM protein ; 30.5 M-1.s-1, 17 µM protein ; 9 M-

1.s-1), EcDosH M95I (113 M-1.s-1) and myoglobin (117 M-1.s-1).

Sup. Mat. 3. EcDosH kinetics of CO binding, after flash photolysis corresponding to the replacement reaction of Met by CO after the µs->ms ligand rebinding phase and stopped-flow mixing (long time of incubation in the unliganded state). Samples were prepared at 15 µM in PBS buffer, 25°C and 0.5 atm. CO final. Sup. Mat. 4. Oxygen (400 µM) replacement by CO (730 µM) for EcDos after photoincubation of the oxy form by flashing off CO (10 s, 10 Hz laser frequency). The insert shows the Soret absorption changes.

Supplementary materials 1

12 13 14 15

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

1e+4

2e+4

3e+4

4e+4

5e+4

6e+4

elution volume (ml)

rela

tive

abs

orba

nce

scal

e

mol

ar m

ass

(g/m

ol)

32 KDa

A

13 14 15 16

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

1e+4

2e+4

3e+4

4e+4

5e+4

6e+4

elution volume (ml)

rela

tive

abso

rban

ce s

cale

mo

lar

ma

ss (

g/m

ol)

17 KDa

32 KDa

B

KCN (M)

0.000 0.002 0.004 0.006 0.008 0.010

Bin

ding

Rat

e (s

-1)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

EcDos

EcDosH 0.3 µM

EcDosH 17 µM

EcDosH M95IMb

Supplementary materials 2

time (s)

0.0 0.2 0.4 0.6 0.8 1.0 1.2

0.0

0.2

0.4

0.6

0.8

1.0

AN

stopped-flow mixing

flash photolysis

Supplementary materials 3

time (s)

0 10 20 30 40 50

0.00

0.02

0.04

0.06

0.08

0.10

O.D

. (4

35-

410

nm

)

wavelenght (nm)

380 400 420 440 460 480

O.D

.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Supplementary materials 4

Marten H. Vos, Ursula Liebl and Laurent KigerChristophe Lechauve, Latifa Bouzhir-Sima, Taku Yamashita, Michael C. Marden,

Sensor Protein Dos from Escherichia coli and Its Isolated Heme DomainHeme Ligand Binding Properties and Intradimer Interactions in the Full-length

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