Structural and Functional Properties of a Single DomainHemoglobin from the Food-borne PathogenCampylobactor jejuni*

Received for publication, May 30, 2007, and in revised form, July 2, 2007 Published, JBC Papers in Press, July 2, 2007, DOI 10.1074/jbc.M704415200

Changyuan Lu‡, Masahiro Mukai§, Yu Lin‡, Guanghui Wu¶, Robert K. Poole¶, and Syun-Ru Yeh‡1

From the ‡Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, the §MitsubishiKagaku Institute of Life Sciences, Minamiooya, Machida, Tokyo 194-8511, Japan, and the ¶Department of Molecular Biology andBiotechnology, The University of Sheffield, Sheffield, S10 2TN United Kingdom

Campylobacter jejuni contains two globins, a truncatedhemoglobin, Ctb, and a single domain hemoglobin, Cgb. Thephysiological function of Ctb remains unclear, whereas Cgb hasbeen linked to NO detoxification. With resonance Raman scat-tering, the iron-histidine stretching mode of Cgb was identifiedat 251 cm�1. This frequency is unusually high, suggesting animidazolate character of the proximal histidine as a result of theH-bonding network linking the catalytic triad involving theF8His, H23Glu, and G5Tyr residues. In the CO-complex, twoconformers were identified with the �C-O/�Fe-CO at 529/1914cm�1 and 492/1963 cm�1. The former is assigned to a “closed”conformation, in which the heme-bound CO is stabilized by theH-bond(s) donated from the B10Tyr-E7Gln residues, whereasthe latter is assigned to an “open” conformer, in which theH-bonding interaction is absent. The presence of the two alter-native conformations demonstrates the plasticity of the proteinmatrix. In the O2-complex, the iron-O2 stretching frequencywas identified at 554 cm�1, which is unusually low, indicatingthat the heme-bound O2 is stabilized by strong H-bond(s)donated by theB10Tyr-E7Gln residues. This scenario is consist-ent with its low O2 off-rate (0.87 s�1). Taken together the datasuggest that the NO-detoxifying activity of Cgb is facilitated bythe imidazolate character of the proximal F8His and the distalpositive polar environment provided by the B10Tyr-E7Gln.Theymayoffer electronic “push” and “pull,” respectively, for theO-O bond cleavage reaction required for the isomerization ofthe presumed peroxynitrite intermediate to the product,nitrate.

Three groups of hemoglobins (Hbs)2 have been identified inmicroorganisms: flavohemoglobins (FHbs), single domain Hbs

(sdHbs), and truncated Hbs (trHbs) as shown in Scheme 1 (1).The FHbs contain a globin domain with a classical three-over-three �-helical structure and an additional flavin-containingreductase domain covalently attached to it (2–6). The sdHbsshare high sequence and structure homology with the globindomain of the FHbs. The trHbs, on the other hand, are muchsmaller; they contain �110–140 amino acid residues andexhibit a two-over-two �-helical structure, which is character-ized by the absence of the A-helix and the presence of anextended loop substituting for the F-helix (7, 8). On the basis ofphylogenetic analysis, the trHbs can be further divided intothree subgroups, trHb-I, trHb-II, and trHb-III (1).The various classes of microbial Hbsmay coexist in the same

organism. For example,Mycobacterium tuberculosis contains atrHb-I (trHbN) and a trHb-II (trHbO) (8), Mycobacteriumavium contains three trHbs, one from each subgroup (1),whereas Campylobacter jejuni contains a trHb-III (Ctb) and asingle domain Hb (Cgb) (9, 10). These findings suggest distinctfunctions for each class of Hb. Despite their functional diver-sity, all microbial Hbs discovered to date contain a highly con-served tyrosine residue at the B10 position and a histidine res-idue at the F8 position (8). The distal histidine at the E7position, which is important in stabilizing heme-bound dioxy-gen in mammalian globins, may be replaced by a variety ofdifferent polar or non-polar residues. Spectroscopic studieshave shown that H-bonding interactions involving the distalpolar residues at the B10, E7, E11, CD1, and/or G8 positionsplay an important role in regulating the ligand binding andfunctional properties of microbial Hbs (1, 6, 8, 10).C. jejuni is a Gram-negative, obligatory microaerophilic bac-

terium. It is present in the gut ofmany food-supply animals andbirds and is one of the leading causes of bacterial gastroenteritisworldwide. Neither Ctb nor Cgb are required for the survival ofthe bacterium in air (11, 12), but theO2 consumption rate of ctbknock-out mutant cells showed a 50% reduction as comparedwith the wild-type cells, suggesting the involvement of Ctb inregulating the flux of O2 into and within the cell (13). Nonethe-less, the role of Ctb as a direct oxygen transporter has been

* This work is supported by National Institutes of Health Grant HL65465 (toS.-R. Y.) and a Biotechnology and Biological Sciences Research Council(BBSRC, United Kingdom) Grant (to R. K. P.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Physiology andBiophysics, Albert Einstein College of Medicine, Bronx, NY 10461. Tel.: 718-430-4234; Fax: 718-430-4230; E-mail: syeh@aecom.yu.edu.

2 The abbreviations used are: Hb, hemoglobin; CCP, cytochrome c peroxi-dase; Cgb, single domain hemoglobin from C. jejuni; Ctb, truncated hemo-globin III from C. jejuni; mCPBA, m-chloroperbenzoic acid; FHb, flavohemo-globin; Hmp, flavohemoglobin of E. coli; Mb, myoglobin; NOD, nitric-oxidedioxygenase; sdHb, single domain hemoglobin; trHb, truncated hemoglo-

bin; trHbC, truncated hemoglobin from green algae C. eugametos; trHbN,truncated hemoglobin I from M. tuberculosis; trHbO, truncated hemoglo-bin II from M. tuberculosis; trHbP, truncated hemoglobin from protozoanP. caudatum; trHbS, truncated hemoglobin from cyanobacterium Synecho-cystis sp. PCC 6803; Vgb, single domain hemoglobin from Vitreoscilla sp;HRP, horseradish peroxidase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 35, pp. 25917–25928, August 31, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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excluded due to its unusually slow O2 association and dissoci-ation rates (9.1� 105M�1 s�1 and 0.0041 s�1, respectively) (10).The expression of Ctb can be induced by NO donors, althoughthe Ctb knock-outmutant ofC. jejuni does not display any sen-sitivity to nitrosative stress (12). On the other hand, resonanceRaman studies show that the distal heme pocket of Ctb resem-bles that of cytochrome c peroxidase (CCP), suggesting a roleforCtb in performing oxygen chemistry (10). In contrast toCtb,the Cgb knock-outmutant ofC. jejuni is hypersensitive to reac-tive nitrogen species; furthermore, the expression of Cgb isstrongly and specifically induced by nitrosative stress (9). Thus,Cgb, like the FHb from Escherichia coli (Hmp), is believed tofunction as a NO dioxygenase (NOD) to protect the bacteriumagainst the toxic effects of NO (9).The structural properties of Cgb, in contrast to its physiolog-

ical role, are relatively unexplored. Here, we have overex-pressed and purified Cgb from E. coli. We have examined thestructural properties of Cgb by using resonance Raman spec-troscopy and investigated its chemical reactivity toward perox-ides and its ligand binding properties by using a stopped-flowsystem.We conclude that the structural and functional charac-teristics of Cgb are distinct from Ctb, consistent with thehypothesis that the two Hbs perform distinct physiologicalfunctions. On the other hand, albeit subtle differences, theproperties of Cgb are comparable to those of Hmp from E. coli,in line with its potential function as a NO dioxygenase.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The cloning, expres-sion, and purification of Cgb are described in detail elsewhere(9). Briefly, the plasmid containing the cgb gene was trans-formed into the BL21 (DE3) strain of E. coli. The starter cul-tures were grown overnight in LB medium supplemented withampicillin (100�g/ml). Starter cultures were then used to inoc-ulate (at 1% v/v) 2-liter baffled flasks containing 500ml of 2xYT(or LB) medium supplemented with 100 �M ampicillin, and 1mM aminolevulinic acid/3 �M FeCl3, or 8.0 �M hemin. Whenthe culture reached �100 Klett units, 0.5 mM isopropyl-1-thio-�-D-galactopyranoside was added to induce the protein expres-sion. After overnight growth at 37 °C, the cells were harvestedand stored at �70 °C until used.For purification, frozen cells were thawed, suspended in 50

mM Tris-HCl buffer (pH 8.0) and disrupted by a sonicator fourtimes (20 s each) in an icy water bath with a 1-min chill inbetween. The cell debris was removed by centrifugation at44,000 � g for 20 min at 4 °C. The supernatant was passedthrough a 30-mlDEAESepharose Fastflow column (Amersham

Biosciences) equilibrated with 50 mM Tris-HCl, pH 8.0, fol-lowed by a 10-ml Butyl-Toyopearl 650S column equilibratedwith 1.5 M ammonium sulfate in 50 mM Tris-HCl, pH 8.0. Theprotein thus collected was concentrated and further purifiedwith a Superdex-200 column (Amersham Biosciences) equili-brated with 0.1 M NaCl in 50 mM Tris-HCl (pH 8.0). Pure Cgbwas stored at �80 °C until used.To avoid cyanide binding to Cgb (vide infra), we used an

alternative purification method, starting with E. coli cells thathad been disrupted by a French pressure cell, instead of a son-icator. This was accompanied by small modification in the sub-sequent purification procedures as follows. The cell debris wasremoved by centrifugation at 16,000 rpm for 30min at 4 °C. Thecrude cell extract was purified with ammonium sulfate frac-tionation, followed by dialysis overnight against 10 mM Tris-HCl buffer with 50 �M EDTA, pH 8.0. The protein was thenpassed through a 400-ml anion exchange column (WhatmanDE 52) equilibrated with 10 mM Tris-HCl buffer with 50 �MEDTA (pH 8.0). It was washed with 0.02 M NaCl in 10 mMTris-HCl buffer and elutedwith 0.05MNaCl in 10mMTris-HClbuffer. The protein thus collectedwas concentratedwith aCen-tricon and stored at �80 °C until used.Preparation of the Various Derivatives of Cgb—For spectro-

scopic studies, Cgb purified after cell disruption by the Frenchpressure cell was treated with 10-fold excess of potassium fer-ricyanide to ensure that the protein was in a homogeneous fer-ric state. The excess of ferricyanide and its reduction productthus generated were removed by a G25 column. The deoxysample was prepared by first flushing the sample with Ar andthen reduced with 5-foldmolar excess of sodium dithionite. Toprepare the CO-bound complexes, 12C16O or 13C18O wasinjected into the reduced Cgb under anaerobic conditions. The16O2-bound derivative was prepared by passing the dithionite-or NADH-reduced protein through a G-25 column under aer-obic conditions to remove the excess of dithionite or NADHand to allow atmospheric oxygen to bind to the protein. The18O2-bound sample was prepared by injecting 18O2 into 16O2-bound protein solution, freshly purged with argon, and allow-ing the spontaneous exchange of the heme-bound 16O2 with18O2. The cyanide complexes were formed by addition of �20-fold excess of KCN or its various isotopic derivatives.CO (Matheson purity) and Ar (99.999%) were obtained from

Tech Air (New York). K13C14N, K12C15N, K13C15N, 18O2, and13C18Owere purchased from Icon (Icon Isotopes, Summit, NJ).All solutions were prepared with deionized (Millipore) water.Unless otherwise indicated, the Cgb protein samples were buff-ered with 50 mM Tris-HCl and 50 �M EDTA at pH 7.4.Optical Absorption Spectroscopy—The optical absorption

spectra were recorded by using a Shimazu spectrophotometer(ShimazuCo., Kyoto, Japan). All spectra were recorded at roomtemperature (�25 °C). The protein concentrations were�1–10 �M.Resonance Raman Spectroscopy—The resonance Raman

measurements were carried out with previously describedinstrumentation (8). Briefly, the output at 413.1 nm from akrypton ion laser (Spectra Physics) was used as the excitationsource, unless otherwise indicated. The laser line was focusedon a quartz cuvette, constantly rotating at �6000 rpm to avoid

SCHEME 1

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photodamage of the sample. The scattered lightwas collected atright angles to the incident beam and focused on the entranceslit (100�m)of a polychromator (Spex,Metuchen,New Jersey),where it was dispersed by a 1200 grooves/mm grating and sub-sequently detected by a liquid nitrogen-cooled charge-coupleddevice (CCD) (Princeton Instruments, Trenton, New Jersey). Ahalographic notch filter (Kaiser, Ann Arbor) was used to elim-inate the Rayleigh scattering. The Raman spectra were cali-brated with indene (Sigma) for the 200–1700 cm�1 spectralregion or acetone/potassium ferrocyanide for the 1700–2300cm�1 spectral region.Optical absorption spectrawere acquiredbefore and after the Raman measurements to ensure the integ-rity of the samples. The Raman datawere processed byGRAMSsoftware (Galactic Industries Corp.).Stopped-flow Measurements—The reactions of Cgb, Hmp,

and horse heart myoglobin (hhMb) with hydrogen peroxideand m-chloroperbenzoic acid were measured at 50 mM phos-phate (pH 7.4) with a �*180 stopped-flow instrument fromApplied Photophysics Inc. (Leatherhead, UK). The final con-

centrations of Hmp and hhMbwere2 and 1 �M, respectively. The finalconcentrations of Cgb were �3 and�2�M for the reactions with hydro-gen peroxide and m-chloroperben-zoic acid, respectively. The reactionkinetics were probed in an opticalabsorption mode with a photodiodearray detector. The reaction kinet-ics was analyzed with the ProK soft-ware from Applied PhotophysicsInc. TheHmp samplewas expressedand isolated as described elsewhere(6), and the hhMb sample wasobtained from Sigma-Aldrich andused without further purification.Hydrogen peroxide and m-chlo-roperbenzoic acid were obtainedfrom Sigma-Aldrich as well.

O2 and CO Dissociation Kinetic Measurements—The liganddissociation kinetic experiments were carried out at 20 °C withsingle wavelength stopped-flow apparatus from Applied Pho-tophysics Inc. In these experiments, �1–3 �M O2-bound Cgbsample (in the presence of 20 �M free O2)or CO-bound Cgbsamples (in the presence of 80�M freeCO) in 50mMTris buffer,pH 7.4 were mixed with high concentrations of CO or NO,respectively. For each measurement, at least two differentligand concentrationswere examined to ensure the reliability ofthe data. The reactions were followed at two different wave-lengths (the Soret maxima of the reactant and product). Thereaction rates were obtained from the fits of 5–6 traces. Thedissociation rate constants were calculated based on Equations1 or 2.

koff,O2 � kobs �1 � kon,O2�O2�/kon,CO�CO�� (Eq. 1)

or

koff,CO � kobs �1 � Kon,CO�CO�/kon,NO�NO�� (Eq. 2)

Here, kobs is the observed ligand-replacement rate.

RESULTS AND DISCUSSIONS

Sequence Alignment—Phylogenetic analysis of Cgb showedthat it shares 42% amino acid identity with Vgb (a sdHb fromVitreoscilla sp) and 33% identity with the globin domain ofHmp (the FHb from E. coli) (9), in contrast to the only �10%identity with spermwhale myoglobin (swMb). As shown in Fig.1, the proximal F8 residue in Cgb is a histidine and the distalB10, E7, E11, and CD1 residues, which have been shown to becritical for ligand-protein interactions in microbial Hbs, areTyr, Gln, Leu, and Phe, respectively. These residues are fullyconserved in Cgb, Vgb, and Hmp.Purification of Cgb—On the basis of the reduced-minus-ox-

idized pyridine-hemochromogen spectrum (14), the isolatedCgb protein contains a noncovalently bound B-type heme (datanot shown). The as-isolated protein prepared with the sonica-tion method exhibits a Soret maximum at 417 nm and a broadvisible band at �542 nm (Fig. 2). To further characterize this

FIGURE 1. Sequence alignment of Cgb with Vgb and the globin domain of Hmp. The hemoglobin sequencedata were obtained from TIGR.

FIGURE 2. Electronic absorbance spectra of Cgb prepared with the soni-cation and the French pressure cell methods. The Cgb samples were pre-pared as discussed in the text.

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species, wemeasured its resonance Raman spectrumwith Soretexcitation at 413.1 nm.Resonance Raman spectroscopy is a powerfulmethod for the

studies of hemeproteins, because excitation in resonance withthe porphyrin �-�* electronic transition selectively enhancesvibrational modes of the heme and its bound ligands, withoutinterference from themodes associatedwith the proteinmatrix(8). The spectrum in the high frequency region (1100–1700cm�1) consists of porphyrin in-plane vibrational modes, whichare markers for the oxidation-state, coordination-state andspin-state of the heme iron (15). On the other hand, the spec-trum in the low frequency region (200–1200 cm�1) containsin-plane and out-of-plane vibrational modes of the porphyrinmacrocycle, as well as modes associated with the ligands andthe peripheral groups of the heme, including propionate andvinyl groups (16).As shown in Fig. 3A, the �2, �3, and �4 modes of the as-iso-

lated form of Cgb are identified at 1584 and 1505 and 1374cm�1, respectively, characteristic of a six-coordinate low-spin(6CLS) heme. No changes in optical and Raman spectra wereobserved upon the treatment of the as-isolated protein withoxidants, such as ammonium persulfate or potassium ferricya-nide, suggesting that the heme iron was in the ferric state. Incontrast, treatment with sodium dithionite led to slow reduc-tion of the heme to a five-coordinate high-spin (5CHS) deriva-tive, as indicated by the appearance of the �3 and �4 modes at1469 and 1353 cm�1, respectively, at the expense of the modesassociated with the 6CLS species (Fig. 3A), consistent with theproposal that the as-isolated protein was in the ferric state. It isnoted that the reduction did not reach completion even after a5-day incubation under anaerobic conditions (Fig. 3A).A number of Hbs, such as neuroglobin, cytoglobin, and

synechocystis Hb, have been found in a 6CLS state, in whichthe distal histidine residue coordinates to the heme iron inthe absence of exogenous ligand (17–20). To test if Cgb, liketheseHbs, is coordinated by an intrinsic amino acid in the ferric

state and if an exogenous cyanideligand can compete with the pre-sumed intrinsic ligand for hemeiron binding, we treated the proteinwith 2 mM potassium cyanide over-night. Interestingly, no spectralchanges were observed. On theother hand, we found that treat-ment with pH 3.6 acetate buffer (50mM), followed by a G25 columnchromatography and dialysisagainst pH 8.0 Tris buffer, causedthe Soret and visible bands to shiftto 398 nm and 501/641 nm, respec-tively, consistent with a 5CHS ferricheme as reported for Hmp and Vgb(3, 21). Moreover, it was found thatthe original spectrum of the as-iso-lated protein (Fig. 2) can be fullyrestored by exposing the acid-treated protein to potassium cya-nide. These data strongly suggest

that the as-isolated protein prepared by the sonication methodis a cyanide-bound ferric derivative of Cgb. To confirm thishypothesis, the low frequency Raman spectrum of the 13C15N-bound protein, generated by incubating the as-isolated proteinwith excess of 13C15N (to allow for the free exchange of thepresumed natural abundant 12C14N pre-bound to the hemeironwith 13C15N), was obtained and subtracted from that of theas-isolated protein without the treatment. The resulting differ-ence spectrum is almost identical to the 12C14N-13C15N differ-ence spectrumof the cyanide-bound protein prepared from the5CHS ferric protein pre-treated with acid as described above(Fig. 3B), confirming that the as-isolated protein is indeed in acyanide-bound ferric state.Because the crude cell lysate from E. coli exhibits the same

spectrum as that of the as-isolated protein, contamination dueto the chromatographic procedures was ruled out. On the otherhand, the presence of cyanide ion has been found in the cellextract from the alga Chlorella vulgaris Beijerinck, when it isdisrupted by sonication but not by the French pressure cell (22).To test if the cyanide found in Cgb is of similar origin, werepeated the protein preparation by disrupting the E. coli cellswith a French pressure cell, instead of a sonicator. As shown inFig. 2, with this new method, the Cgb protein was isolated in aform with a Soret maximum at 405 nm and visible bands at�538, 576, and 638 nm. Further spectral analysis indicates thatthe protein is in a mixture of the oxygen-bound ferrous andexogenous ligand-free ferric states, because the spectrum canbe perfectly simulated by a linear combination of their corre-sponding spectra shown in Fig. 4B. Subsequent treatment of theCgb sample with an oxidant, potassium ferricyanide, convertedthe proteinmixture to a homogeneous ferric 5CHS specieswithSoret and visible bands at 397 nm and 501/641 nm, respectively(Fig. 4B), identical to the spectrum of the acid-treated proteinsample preparedwith the sonicationmethod. The data confirmthat the exogenous cyanide ligand is an artifact derived from thesonication procedure.

FIGURE 3. Resonance Raman spectra of the as-isolated Cgb prepared with the sonication method as afunction of time following the treatment with sodium dithionite under anaerobic conditions (A), andthe comparison between the cyanide-bound protein with and without acid-treatment (B). In B, the topthree traces are the spectra of the as-isolated protein in the absence and presence of K13C15N and theirdifference spectrum; the bottom trace is the 12C14N-13C15N difference spectrum of the as-isolated proteinpre-treated with acid as described in the text.

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In addition to the alga C. vulgaris Beijerinck, several bacteriaand plants have been described as being cyanogenic, i.e. able toform hydrocyanic acid (HCN) (23, 24), although the source ofcyanide remains unclear. Nonetheless, it has been suggestedthat cyanide is produced by oxidative reactions of amino acids(such as glycine or histidine) and, more interestingly, it wasfound that cyanide production is stimulated by the addition ofperoxidases (22, 24). Similarly, it has been reported that endog-enous cyanide can be generated in white blood cells and neuro-nal tissues, via a mechanism involving peroxidase activity (25).The fact that Cgb exhibits a peroxidase-like heme active site(vide infra) implies that the overexpression ofCgb in E. colimayfacilitate cyanide generation, accounting for the presence ofcyanide in the Cgb samples.For the ensuing studies, the Cgb samples were prepared with

the French pressure cell protocol with minor modifications as

described under “Experimental Pro-cedures.” In addition, all the puri-fied protein samples were oxidizedby potassium ferricyanide and fur-ther purified with a G25 columnprior to use to ensure the homoge-neity of the samples. To reveal thestructural properties of Cgb, theexogeneous ligand-free ferric andferrous derivatives as well as theircyanide and CO/O2 complexes,respectively, were studied with res-onance Raman spectroscopy.Exogenous Ligand-free Deriva-

tives—The �2, �3, and �4 modes ofthe ferric Cgb were identified at1570, 1493, and 1371 cm�1, respec-tively, typical for a 5CHS heme (Fig.4A). The ferric protein can be easilyreduced to a 5CHS deoxy form with

dithionite or NADH, as indicated by the shift of the Soret andvisible bands from 397 and 501/641 nm to 432 and 555 nm,respectively (Fig. 4B). The resonance Raman spectrum of thedeoxy form thus obtained shows �3 and �4 modes at 1470 and1353 cm�1, respectively (Fig. 4A). The 1604 cm�1 band isassigned to the �10 mode, a core size marker involving theCa–Cm stretch, whereas the 1620 and 1627 cm�1 modes areassigned to the two �C�C stretchingmodes of the 2- and 4-vinylgroups (26). In the low frequency region (Fig. 5), the 361/372cm�1 and 417 cm�1 modes are assigned to the out-of-planepropionate bendingmodes, �(C�CcCd), and an in-plane vinylbending mode, �(C�CaCb), respectively. The strong band at251 cm�1 is assigned to the proximal iron-His stretchingmode (�Fe-His), which is also present in the spectrum of thephotoproduct of the CO-complex, when CO is photolyzedfrom the heme iron.The frequency of the �Fe-His mode of Cgb, like that of Hmp

(244 cm�1) (6), is much higher than that of most globins (typi-cally 200–220 cm�1) (8). The high frequency of the �Fe-Hismode in Hmp has been attributed to the imidazolate characterof the proximal histidine due to the presence of a strongH-bonding network connecting the proximal F8His-H23Glu-G5Tyr residues making up the catalytic triad (6), as shown inFig. 6A (27). The same H-bonding network is present in Vgb(Fig. 6B) (28), which also exhibits relatively high �Fe-His fre-quency (252 cm�1).3 Because the H23Glu and G5Tyr residuesare conserved in Cgb (Fig. 1), the high �Fe-His frequency foundin Cgb is attributed to the same origin.The ferric Cgb, like Hmp and Vgb, exhibits a 5CHS configu-

ration, indicating that the distal ligand binding site of the hemeis unoccupied, in contrast to the more commonly observedwater-bound ferric heme in other globins (8). Like that pro-posed for Hmp (6), the absence of a water molecule bound tothe heme iron of Cgb can be ascribed to the strong proximalFe-His bond (as reflected by the high �Fe-His frequency), which

3 S. Yeh, submitted manuscript.

FIGURE 4. Resonance Raman spectra (A) and electronic absorbance spectra (B) of the various derivativesof Cgb at pH 7.4. The peak maxima in B are as follows: Fe2, 432 and 555 nm; Fe3, 397, 501, and 641 nm;Fe2-CO, 419, 538, and 565 nm; and Fe2-O2, 412, 541, and 576 nm.

FIGURE 5. Resonance Raman spectra of the deoxy derivative of Cgb in thelow-frequency region under differing pH conditions. Inset, a plot of thepercentage of the area under the 251 cm�1 peak versus the total area underthe 229 and 251 cm�1 peaks as a function of pH.

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pulls the heme iron out of the porphyrin plane, thereby pre-venting the coordination of the water to the heme iron due tothe repulsive force between the water and the pyrrole nitrogenatoms of the porphyrin ring.As shown in Fig. 5, lowering the pH of the ferrous protein

induced the development of a shoulder on the low wavelengthside of the �Fe-His mode at�229 cm�1 at the expense of the 251cm�1 band, although the high frequency Raman spectra showthe heme remained in its 5CHS configuration (data not shown).The inset in Fig. 5 shows the percentage of the area under the251 cm�1 peak versus the total area under the 251 and 229 cm�1

peaks as a function of pH. Because the protein is not stableunder pH conditions lower than 4, it is not possible to deter-mine the exact pka associated with this transition. Nonetheless,the data indicate a pKa lower than 6, suggesting that thepH-induced transition is a result of the protonation of anacidic residue, possibly the H23Glu, and the protonationleads to the concurrent weakening of the Fe-His bond (asreflected by a lower �Fe-His frequency) due to the disruption

of the H-bonding network linkingthe proximal catalytic triad. Inaddition to the modification in the�Fe-His mode, two modes at 377and 392 cm�1 are significantlyenhanced at low pH, which areassociated with small changes inseveral other heme modes in the330–340 and 700–850 cm�1

spectral regions, reflecting thepH-induced structural perturba-tions in the porphyrin ring as wellas the two propionate groups ofthe heme. On the other hand,disturbance of the proximal cata-lytic triad by the mutation of theG5Tyr to Phe caused the proteinto convert to a 6CLS form due tothe coordination of an intrinsicamino acid to the heme iron (datanot shown). Taken together the

data indicate that H23Glu and G5Tyr in Cgb are importantin maintaining the integrity of the protein matrix, as well asin modulating the electron donating capability of the proxi-mal histidine heme ligand, which is important in providingelectronic push for promoting the isomerization of the per-oxynitrite intermediate to nitrate as suggested for Hmp (6).CO-bound Ferrous Complex—The CO-derivative of Cgb dis-

plays a narrow Soret band at 419 nm and �/� bands at 565/538nm (Fig. 4B). In the resonance Raman spectrum, the �2, �3, and�4 modes are identified at 1584, 1498, and 1372 cm�1, respec-tively (Fig. 4A), characteristic for a 6CLS heme. In the low fre-quency region of the resonance Raman spectrum (Fig. 7), thetwo peaks at 492 and 529 cm�1 shift to 482 and 515 cm�1,respectively, upon the substitution of 12C16O with 13C18O.They are assigned to two Fe-CO stretching modes (�Fe-CO).Similarly, the two peaks at 1914 and 1963 cm�1 in the highfrequency region are assigned to two C-O stretching modes(�C-O), as they shift to 1828 and 1873 cm�1, respectively, uponthe substitution of 12C16O with 13C18O.The Fe-C-O moiety in hemeproteins normally exists in the

following two extreme resonance structures due to the � back-bonding from the iron to the CO, which donates electron den-sity from the d� orbital of the iron back to the �* orbital of theCO in Equation 3.

L™Fe� ™C§O� (I) 7 L™Fe¢C¢O �II� (Eq. 3)

Here, L represents the proximal ligand of the heme iron, whichis a histidine in the case of globins. In general, a positive polarenvironment destabilizes form (I) and facilitates the � back-bonding interaction, leading to a stronger Fe-CO bond and aweaker C-O bond. On this basis, the frequency of the �Fe-COmode is typically inversely correlated with that of the �C-Omode in a linear fashion (8, 29, 30). The inverse correlation lineis sensitive to the proximal ligand, such that the line for histi-dine coordinated species is distinct from that of thiolate-coor-dinated species and five-coordinate CO adducts. Because of its

FIGURE 6. The active site structure of (A) Hmp (PDB entry: 1GVH) and (B) Vgb (PDB entry: 1VHB). The B andE helices are labeled in green and yellow, respectively. The dotted magenta lines indicate the H-bonding inter-actions. The dotted blue line in B indicates the missing fragment (residues 44 –52) in Vgb.

FIGURE 7. Resonance Raman spectra of the 12C16O and 13C18O-coordi-nated forms of Cgb and the difference spectrum between them.

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sensitivity to the identity of the proximal heme ligand, as well asthe distal environment, CO has beenwidely used as a structuralprobe for hemeproteins.On the basis of the inverse correlation, the �C-O and �Fe-CO of

Cgb at 529 and 1914 cm�1 are assigned to a “closed” conformer,in which the heme-bound CO is stabilized by H-bond(s)donated from the B10Tyr-E7Gln pair, whereas those at 492 and1963 cm�1 are assigned to an “open” conformer, in which theH-bonding interaction is absent. The data points associatedwith the two conformers fall on the histidine correlation curve(Fig. 8), confirming the assignment of the proximal ligand as ahistidine.In general, the CO-derivatives of microbial Hbs can be

divided into two groups: (I) those adopting two conformerswith �Fe-CO at �490 and �530 cm�1 for the open and closedconformers, respectively, and (II) those displaying a single fixedconformation with �Fe-CO varying from �490 to 525 cm�1 (8,10). Remarkably, all the Hbs belonging to the first group, suchas Cgb, Hmp, and trHbN, have been implicated in performingNOD reaction in vivo to protect the corresponding organisms

fromnitrosative stress (6, 31, 32). TheNOD reaction is believedto proceed via the following mechanism in Equation 4.

Fe2™O™O � �N¢O3 �Fe3™O™O™NO��3 Fe3 � NO3�

(Eq. 4)

Here the heme-bound peroxynitrite (Fe3-OONO�) is a pos-tulated intermediate, which isomerizes to form the product,nitrate (33). The ability of Cgb, Hmp, and trHbN to adopt aclosed conformer with high electrostatic potential surroundingthe heme ligand presumably is important for providing an elec-tronic pull, in addition to the electronic push offered by theproximal imidazolate ligand, for facilitating the isomerizationof the peroxynitrite intermediate to nitrate as illustrated in Fig.9B. On the other hand, the conformational flexibility of theseHbs, allowing them to adopt an alternative open conformer, inwhich no direct electrostatic interaction can take place betweenthe ligand and the proteinmatrix surrounding it, may be crucialfor the efficient release of the nitrate product during the multi-ple turnover of the NOD reaction.It is noticeable that the relative population of the open con-

former with respect to the closed conformer in the CO deriva-tive of Cgb is pH-insensitive, in contrast to that observed inHmpwhich exhibits a pKa of�8.3 as shown in Fig. 9A, suggest-ing that, despite their similarities, the specific intramolecularinteractions distinguishing the open conformer from the closedconformer in these two Hbs are distinct. The presence of thePro residue at the E8 position in Cgb, but not in Hmp (Fig. 1),may account for the absence of the pH dependence in the for-mer, because the Pro ring could exhibit steric effects that alterthe H-bonding interaction between the heme-bound CO andthe B10Tyr-E7Gln pair.In addition to the �Fe-CO and �C-O modes, a bending mode

(�Fe-C-O) is identified at 583 cm�1, which shifts to 560 cm�1

upon the substitution of 12C16O with 13C18O (Fig. 7). Recently,Das et al. (34) recognized inverse correlations between �Fe-C-Oand �Fe-CO (�C-O) in hemeproteins with a histidine or cysteineas the proximal heme ligand. Because the �Fe-C-O mode is typ-ically enhanced when the Fe-C-O is in a bent form (35), thebending mode of Cgb at 583 cm�1 is assigned to the closedconformerwith �C-O at 1914 cm�1, inwhich the Fe-C-Omoietyis presumably forced to be bent due to the distal H-bondinginteractions exerted on it. This scenario is consistent with thefact that the Cgb data with �Fe-C-O/�C-O at 583/1914 cm�1 lies

FIGURE 8. The �Fe-CO versus �C-O inverse correlation line for the variousmicrobial Hbs (A), and the �Fe-CO versus �C-O inverse correlation line forthe various hemeproteins (B). The data shown in A are taken from Table 1.The data shown in B are taken from the literature (6, 8, 10, 30, 34, 52–55). Lstands for the proximal ligand of the heme iron.

FIGURE 9. A plot of the ratio of the peak area of the �Fe-CO mode at 492cm�1 with respect to the sum of the peak areas of the �Fe-CO mode at 492and 529 cm�1 as a function of pH (A), and the “push-pull” model for theactivation of the peroxynitrite-bound intermediate of Cgb during theNO dioxygenase reaction. The Hmp data in A are taken from Ref. 6.

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on the �Fe-C-O-�C-O inverse correlation line for hemeproteinswith a histidine as the proximal ligand (Fig. 8B).Oxygen-bound Complex—The oxy complex of Cgb is stable

for several months at�20 °C, in contrast to that of Hmp, whichexists only transiently (3, 36). It exhibits a Soret maximum at412 nm and �/� bands at 576/541 nm (Fig. 4B). In the lowfrequency Raman spectrum, themode at 554 cm�1 shifts to 527cm�1 upon the substitution of 16O2 with 18O2 (Fig. 10). It isassigned to the Fe-O2 stretching mode (�Fe-O2), because theisotopic shift is consistent with that expected for a Fe-O2

diatomic oscillator.On the other hand, although the �O-Omodeis expected to be in the �1120–1200 cm�1 region (10, 37), thepositive and negative peaks at �1130 cm�1 in the 16O2-18O2isotope difference spectrum is assigned as an artifact becausethe frequency shift is much smaller than that expected for anO-O oscillator.The frequency of the �Fe-O2 at 554 cm�1 is much lower than

that of mammalian globins (�570 cm�1), but is comparable tothat of most other microbial Hbs as listed in Table 1 (8, 37). Inmicrobial Hbs, the low �Fe-O2 frequencies have been attributedto the H-bonding interactions between the distal residues andthe proximal oxygen atom of the heme-bound dioxygen (10,38). Accordingly, the low �Fe-O2 frequency of Cgb is attributedto a similar origin, i.e. the H-bonding interaction between thedistal B10Tyr-E7Gln pair and the proximal oxygen atom of theheme-bound dioxygen. This unique distal H-bonding interac-tion is presumably critical for the isomerization of the per-oxynitrite intermediate to nitrate during the NOD reaction asillustrated in Fig. 9B.Cyanide-bound Ferric Complex—Although Fe3-CN and

Fe2-CO are isoelectronic, their chemical properties are dis-tinct. As illustrated in Equation 3, the Fe2-CO bond is sensi-tive to the �-bonding interactions; in contrast, the Fe3-CNbond is dominated by the -bond donated from the 4�* and 5�molecular orbitals of CN� to the dz2 orbital of the heme iron(39). Consequently, Fe3-CN is more flexible than Fe2-COand they respond differently to steric and electrostatic influ-ences exerted by the distal protein matrix surrounding theligand in hemeproteins. The higher flexibility of the Fe-C-Nmoiety is consistent with its lower bending frequency (�350–460 cm�1) with respect to that of the Fe-C-O moiety (�560–590 cm�1) (34, 40). Cyanide-bound ferric complexes ofhemeproteins can adopt either “linear” or “bent” conforma-

FIGURE 10. Resonance Raman spectra of the 16O2 and 18O2-coordinatedforms of Cgb, and the difference spectrum between them.

TABLE 1The Raman and kinetic data of Cgb and other related hemeproteins

Ferrica �Fe-His �Fe-CO �Fe-O2 �O-O Kon,CO koff,CO kon,O2 koff,O2

cm�1 cm�1 cm�1 cm�1 �M�1s�1 s�1 �M�1s�1 s�1

sdHb Cgb 5CHS 251 492, 529 554 NDb 45c 0.024 150c 0.870.19

Vgb 5CHS 252d 31d 200p 4.2p0.66 0.15

FHb Hmp 5CHS 244e 494, 535e 22f 0.057f 38f 0.44 f

1.4 0.018trHbs trHbN 6CHSLS 226g 500, 534g 562g 1139d 5.5d 0.005h 71d 0.16d

trHbO 6CHSLS 226i 525i 564i 1143i 0.01d 0.0015j 0.13d 0.0014 j

0.0041 0.0058Ctb 6CHSLS 226k 515k 542l 1132l 0.064d 0.0048d 0.91l 0.0041l

HRP 5CHS 244m 541, 519nCCP 5CHS 248m 505, 537mswMb 6CHSLS 220o 508o 569o ND 0.51j 0.019 14j 12j

a The 6C, 5C, HS, and LS stand for six coordinate, five coordinate, high spin, and low spin heme iron, respectively.b ND, not detected.c Ref. 49.d To be published.e Ref. 6.f Ref. 46.g Ref. 38.h Ref. 32.i Ref. 56.j Ref. 57.k Ref. 11.l Ref. 10.m Ref. 58.n Ref. 59.o Ref. 8.p Ref. 60.

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tions. Moreover, in the “linear” conformation, the �Fe-CN fre-quencies of hemeproteins with histidine as the proximal ligandare typically higher than those of hemeproteins with a cysteineor tyrosine as the proximal ligand (e.g.P450s or catalase, respec-tively), because cyanide competes with the proximal ligand forthe -bonding with the dz2 orbital of the heme iron.

To examine the cyanide-associated vibrational modes ofCgb, the resonance Raman spectra of the ferric derivative ofCgb were obtained in the presence of 12C14N, 13C14N, 12C15Nor 13C15N, as shown in Fig. 11A. The various isotope differencespectra shown in Fig. 11B exhibit similar patterns as thatreported for the cyanide derivatives of catalases and peroxi-dases (40–43). Accordingly, we assigned the bands at 440 and403 cm�1 to the Fe-CN stretching (�Fe-CN) and bending modes(�Fe-CN), respectively, of a linear Fe-C-N moiety, whereas thebands at 353 and 417 cm�1 are assigned to the �Fe-CN and�Fe-CN modes, respectively, of a bent Fe-C-N moiety.An additional cyanide isotope-sensitive mode found at 314

cm�1 (Fig. 11B) is tentatively assigned to the Fe-His stretchingmode (�Fe-His). It is important to note that, although the �Fe-Hismode is typically active only in the spectrum of a 5CHS ferrousheme with the iron located out of the heme plane, it has beenidentified in various 6C heme-proteins. The sensitivity of thismode to the cyanide isotope substitution is presumably due toits coupling to the distal Fe-CN stretching and/or bendingmodes. A similar assignment has been made for a 309 cm�1

band in the cyanide complex of the Hb from Chironomus,which exhibits sensitivity to both CN and iron isotope substi-tution (44). Likewise, the bands at 315 and 311 cm�1 in thecyanide complexes of the ChlamydomonasHb and lactoperox-idase, respectively, were assigned to the same origin (40, 45).The cyanide derivatives of all the globins reported to date

exhibit only a linear conformer; in addition, their �Fe-CN fre-quencies are typically located at �452–458 cm�1 (40), whicharemore than 10 cm�1 higher than that of the linear conformerof Cgb reported here (440 cm�1). On the other hand, mostperoxidases, like Cgb, display both linear and bent conformers.These data indicate that, unlike conventional globins, Cgb has adistal pocket similar to peroxidases, in which the distalH-bonding interaction involving the B10Tyr-E7Gln residues

weakens the Fe-CN bond and causes it to adopt an additionalbent conformer.ComparisonwithHmpandVgb—The resonance Raman data

presented here show that Cgb shares two common structuralfeatures with Hmp and Vgb: (I) a proximal histidine with imi-dazolate character resulting from the conserved F8His-H23Glu-G5Tyr catalytic triad, as indicated by the unusuallyhigh proximal iron-histidine stretching frequency at 251 cm�1,as well as the 5CHS configuration of the ferric protein, and (II)a polar distal environment with B10Tyr-E7Gln formingH-bond(s) with the heme-bound CO in the CO complex, asindicated by the presence of the closed conformer with high�Fe-CO at 529 cm�1, and the plasticity of the protein matrix, asindicated by its ability to exist in an alternative open conformerwith high �Fe-CO at 492 cm�1.Despite their similarities, Vgb exhibits structural signatures

distinctive from Hmp on the basis of the crystal structures oftheir exogenous ligand-free ferric derivatives shown in Fig. 6: (I)the peptide segment connecting the B and E helices (aminoacids 44–52) is disordered and unresolved; (II) the E7 to E11region of the peptide adopts a coil-like structure, instead of ahelical turn as that observed in Hmp; and (III) the side chaingroup of the E7Gln protrudes into the solvent, instead of point-ing toward the heme propionate group as observed in Hmp. Inany case, on the basis of the crystallographic data, a structuraltransition must take place upon ligand binding to Vgb andHmp, perhaps Cgb as well, to enable the interaction betweenheme-bound ligand and the B10Tyr-E7Gln pair, as indicated bythe resonance Raman data. This type of ligand-induced struc-tural transition is plausibly facilitated by the plasticity of theprotein matrix of these Hbs. Intriguingly, several properties ofCgb also set it apart fromHmp: (I) the relative population of theopen conformerwith respect to the closed conformer in theCOderivative of Cgb is pH-insensitive, whereas that inHmp exhib-its a pKa of �8.3 (Fig. 9A), (II) the O2-complex of Cgb is stable,whereas that of Hmp spontaneously auto-oxidizes to the ferricstate (3, 46). Taken together these data suggest that the specificstructural elements controlling ligand protein interactions inCgb, Vgb, and Hmp are distinct. It is noteworthy that theE7-E11 sequence, QPKAL, of Cgb (Fig. 1) is identical to that ofVgb, but is different from the QREAL sequence in Hmp. Thesequence difference, especially the replacement of the E8Pro inCgb/Vgb with the E8Arg in Hmp, may be at least partiallyresponsible for the observed differences between Cgb/Vgb andHmp.Reactivity toward Peroxides—The imidazolate character of

the proximal histidine ligand and the positive polar distal ligandenvironment of Cgb revealed by the resonance Raman datareported here are similar to those of peroxidases. In peroxi-dases, these structural characteristics have been shown to beimportant in catalyzing the O-O bond cleavage reaction ofheme-bound peroxides, on the basis of the well-known “push-pull” model (47) as that illustrated in Fig. 9B. On this basis, wesought to investigate the peroxidase activity of Cgb by examin-ing its reactionwith hydrogen peroxide. For this work, the reac-tion was initiated by mixing Cgb with at least 5-fold excess ofH2O2 in a stopped-flow instrument; the initial decay kineticsof the ligand-free ferric protein were monitored as a function

FIGURE 11. Resonance Raman spectra of the various CN isotope-coordi-nated forms of Cgb (A) and their difference spectra (B).

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of time following the mixing. Similar reactions were carriedout with hhMb and Hmp as comparisons.To our surprise, both Cgb and Hmp exhibit lower reactivity

toward H2O2 as compared with hhMb. As shown in Fig. 12A,the apparent rate constants for all three globins vary linearlywith the concentration of H2O2, and the bimolecular rate con-stants calculated based on the slopes are 95, 18, and 292M�1 s�1

for Cgb, Hmp, and hhMb, respectively. Nonetheless, the 5-foldhigher activity of Cgb as compared with Hmp is in line with thestructural differences manifested in the resonance Raman datashown in Fig. 9A.To further examine the peroxidase activity of the three glo-

bins, their reactions with an organic peroxide, m-chloroper-benzoic acid (mCPBA), were investigated. Intriguingly, thereactivities of Cgb and Hmp toward mCPBA are comparable,both of which are greatly enhanced as compared with theirreactions with H2O2. The bimolecular rate constants of Cgb,Hmp, and hhMb, calculated based on the data shown in Fig.12B are 4.1 � 105, 4.9 � 105, and 7.9 � 103 M�1 s�1, respec-tively. The much higher reactivities of Cgb and Hmp towardmCPBA as compared with hhMb, demonstrate their potentialfunction as organic peroxidases.

The high reactivity of Hmp toward organic peroxides hasbeen reported for its ferrous derivative, but not the ferric deriv-ative (48). On this basis, it was proposed thatHmp is involved inthe repair of oxidatively damaged membrane lipids due to oxi-dative stress. Although the peroxidase activities of Cgb andHmp discovered in this work are distinct from that reported forthe ferrous derivative of Hmp, as the active species is a ferricheme, the data underscore the scenario that the active site ofCgb and Hmp are optimized to perform oxygen chemistry,rather than oxygen delivery.Ligand Binding Properties—It has been reported that the O2

association reaction of Cgb follows single exponential kineticswith a rate constant of 150 �M�1 s�1, whereas the CO associa-tion reaction follows bi-exponential kinetics with rate con-stants of 45 and 0.19 �M�1 s�1 (49). The dissociation rate con-stant of O2 is determined to be 0.87 s�1 in this work, as shownin Fig. 13A. It is similar to that reported in the literature (49).Onthe other hand the dissociation rate constant of CO is deter-mined to be 0.024 s�1 (Fig. 13B), which is 17-fold slower thanthat reported in the literature (49). To examine if the fasterliterature value is a result of a common artifact due to the pho-todissociation of CO by the probing light source during thestopped-flow kinetic measurements, we repeated the samemeasurements as a function of light intensity. We found that

FIGURE 12. The reaction rates of the ferric derivatives of Cgb, Hmp, andhhMb toward H2O2 (A) and mCPBA (B). The initial decay rate constants wereobtained by spectral fit of the time-resolved data with the ProK software fromApplied Photophysics Inc.

FIGURE 13. The kinetic traces for the O2 (A) and CO (B) dissociation reac-tions of Cgb as compared with hhMb (dotted traces). The solid traces in Bare the kinetic traces for the CO dissociation reaction of Cgb with decreasingprobing light intensity as indicated by the arrow.

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under our experimental conditions, decreasing the light inten-sity does not affect the rate of the reaction, whereas increasingthe light intensity causes significant increase in the rate (Fig.13B), confirming that the faster rate reported in the literature isan artifact due to the photodissociation of CO.As listed in Table 1, the on-rates of O2 and CO in Cgb are

�10- and 90-fold faster than swMb, respectively. On the otherhand, the off-rate of O2 in Cgb ismore than 13-fold slower thanswMb, whereas that of CO is similar to swMb (Fig. 13). In glo-bins, heme-bounddioxygen is typically stabilized by distal polarinteractions. Accordingly, a linear correlation betweenlog(koff,O2) and the electrostatic potential of the heme pocket(as reported by �C-O or �Fe-CO) have been recognized in swMband soybean LegHb derivatives (50, 51). In Fig. 14A, we plot thelog(koff,O2) of the various microbial Hbs along with those ofswMb derivatives as a function of �Fe-CO. It is important to notethat for Cgb, Hmp, and trHbN only the �Fe-CO associated withthe closed conformers are used for the plot, since the O2-com-plexes are in a closed state as indicated by the relatively low�Fe-O2 as discussed earlier(Fig. 10) (10). Intriguingly, all theHbs,which have been implicated in performing theNOD reaction invivo, including Cgb, Hmp, and trHbN, are located in the rightlower corner of the swMbcorrelation line; on the other hand, allthe trHbs, except trHbN and trHbP, sit on a separate correla-tion line. The significantly slower O2 dissociation rate con-

stants of the trHbs and its lower sensitivity to the electrostaticpotential of the distal environment surrounding the hemeligand (as indicated by the smaller slope of the inverse correla-tion line) manifest the unique chemical nature of the Fe-O-Omoiety in trHb-type of globins. On the other hand, the slowerO2 dissociation rate constants of Cgb/Hmp and trHbN withrespect to swMb demonstrate the importance of the distalB10Tyr-E7Gln and B10Tyr-E11Gln residues, respectively, inregulating the ligand binding properties of these three Hbs.As shown in Fig. 14B, the O2 and CO on-rate of microbial

Hbs, which span more than 4 orders of magnitude, are alsoinversely correlated with the frequency of the �Fe-CO mode,suggesting that ligand entry in microbial Hbs is somehow reg-ulated by the electrostatic potential of the heme ligand bindingpocket. Here only the open conformers of Cgb, Hmp, andtrHbN are considered, assuming that the open and closed con-formers are in a thermal equilibrium and only the open confor-mation can uptake ligands. It is noticeable that Cgb, like Hmpand trHbN, is located in the upper left corner of the inverselycorrelation lines; all have fast on-rates. Because theO2-complexis the key derivative of Cgb that detoxifies NO as illustrated inEquation 4, the fast on-rate of O2, as well as its slow off-rate, isexpected to be critical for Cgb to execute the NOD reactionefficiently, by sequestering O2 under O2-limiting conditions(which might be particularly pertinent for this microaerophilicbacterium) and by avoiding the energy wasting O2 dissociationreaction.

CONCLUSIONS

In vivo studies have suggested that Cgb function as a NOD toprotect C. jejuni against the toxic effects of NO (9). In line withthis hypothesis, here we show that the architecture of Cgb ischaracteristic of those proteins that perform oxygen chemistryrather than oxygen transport. Our resonance Raman data showthat Cgb is characterized by having a proximal histidine withstrong imidazolate character, due to the presence of theH-bonding network linking the proximal catalytic triad, F8His-H23Glu-G5Tyr, and a polar distal pocket, due to the presenceof the B10Tyr-E7Gln pair, whichmay offer electronic push andelectronic pull, respectively, for facilitating the isomerization ofthe heme-bound peroxynitrite intermediate to nitrate duringtheNOdioxygenase reaction. The plasticity of the heme pocketof Cgb, on the other hand, may be important for the efficientmultiple turnover of the NOD reaction by facilitating therelease of the product nitrate.

Acknowledgment—We thank Dr. Denis L. Rousseau for many invalu-able discussions.

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A Single Domain Hemoglobin from C. jejuni

25928 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 35 • AUGUST 31, 2007

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YehChangyuan Lu, Masahiro Mukai, Yu Lin, Guanghui Wu, Robert K. Poole and Syun-Ru

Campylobactor jejuniFood-borne Pathogen Structural and Functional Properties of a Single Domain Hemoglobin from the

doi: 10.1074/jbc.M704415200 originally published online July 2, 20072007, 282:25917-25928.J. Biol. Chem. 

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