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J Biol Chem, Vol. 275, Issue 3, 1679-1684, January 21, 2000

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis
STABILIZATION OF HEME LIGANDS BY A DISTAL TYROSINE RESIDUE^*

Syun-Ru Yeh^§, Manon Couture^¶, Yannick Ouellet^¶, Michel Guertin^¶, and Denis L. Rousseau

From the  Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461 and the ^¶ Department of Biochemistry, Faculty of Sciences and Engineering, Laval University, Quebec, G1K 7P4, Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O₂, CO, and OH derivatives of the wild type protein and the B10 Tyr  Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Invertebrate, plant, and bacterial hemoglobins have been the topic of many investigations in recent years in view of their peculiar physiological roles (1). The homodimeric hemoglobin from the aerobic bacterium, Vitreoscilla, is transcribed under oxygen-limiting growth conditions (2, 3). It is postulated that the protein acts as an oxygen carrier under hypoxic conditions and facilitates oxygen diffusion to the terminal oxidase complex. The hemoglobin in the eukaryotic green alga, Chlamydomonas eugametos, is expressed in response to activation of photosynthesis (4, 5). It is proposed to be involved with the photosynthetic electron transfer chains. The leghemoglobins discovered in plants function as oxygen scavengers, preventing the oxidation of the O₂-sensitive nitrogen-fixing machinery of the symbiotic Rhizobium bacteroids (6). In Escherichia coli and Salmonella typhimurium, a two-domain flavo-hemoglobin is involved in detoxification of nitric oxide (7-9). Despite this wide array of the physiological functions, all the hemoglobins discovered so far are made of myoglobin-like subunits with a three-over-three -helical sandwich motif (globin fold) (1).

Recently, we expressed and characterized a hemoglobin (HbN)¹ from Mycobacterium tuberculosis that adds a new twist to the scope of the properties of invertebrate hemoglobins (10). HbN is a homodimeric protein. It displays extremely high oxygen binding affinity and cooperativity, with a Hill coefficient of approximately 2. The sequence alignment with hemoglobins from alga, protozoa, and cyanobacteria suggests that the proximal ligand is His, the distal ligand at the E7 position is Leu, and that at the B10 position is Tyr. To examine the structure of the heme pocket and thereby infer the function of this hemoglobin, we have measured the resonance Raman spectra of the wild type protein and the Tyr  Leu and Tyr  Phe mutants under a variety of oxidation and ligand binding states. The resonance Raman spectra reveal distal features that are unique among the hemoglobins and demonstrate the essential role of B10 Tyr in stabilizing the heme-bound ligands.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recombinant M. tuberculosis HbN was cloned, expressed, and purified to near homogeneity as described elsewhere (10). The single amino acid substitution mutants of HbN (Y33L and Y33F) were prepared as described previously (10). The protein was buffered at the desired pH with 50 mM Tris and CAPS at pH 7.5 and 10.5, respectively. For all of the experiments reported here, the protein concentration was 50 µM. The Raman measurements were made with previously described instrumentation (11). The output at 406.7 nm from a krypton ion laser (Spectra Physics) was focused to an ~30 µm spot (laser power ~2 mW) on a rotating cell to prevent photo-damage to the sample. The acquisition time was about 30 min for each spectrum. The scattered light was collected at right angles to the incident beam and focused on the entrance slit of a 1.25 m polychromator (Spex) where it was dispersed and then detected by a charge-coupled device camera (Princeton Instruments).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The general characteristics of resonance Raman spectra of heme proteins are well established (11, 12). In the high frequency region of the spectrum between 1300 and 1700 cm¹, the oxidation state, spin state, and the axial coordination state of the iron at the center of the heme can be characterized. In particular, the ₂ mode, in the region between 1550 and 1600 cm¹, is sensitive to the iron spin state. The line in the 1475-1520 cm¹ region, assigned as the ₃ mode, is sensitive to both the axial coordination and spin state of the iron. The strong line between 1350 and 1400 cm¹, assigned as the ₄ mode, is sensitive to the -electron density of the porphyrin macrocycle and, therefore, the oxidation state of the iron. The frequency and intensity of these Raman lines are further modulated by the protein environment surrounding the heme and, therefore, provide useful structural information on heme proteins. In the low frequency region of the spectrum between 200 and 800 cm¹, the specific axial ligands coordinated to the prosthetic heme group can be identified by detecting iron-ligand stretching modes.

Ligand-free Ferrous HbN-- The high frequency resonance Raman spectrum of the wild type ferrous deoxy form of HbN, shown in Fig. 1A, displays a typical five-coordinate high spin pattern with the electron density line (₄) appearing at 1356 cm¹ and the strong spin/coordination sensitive line (₃) appearing at 1471 cm¹. In the low frequency region, shown in Fig. 2A, a strong line is detected at 226 cm¹ that is assigned as the iron-histidine (FeHis) stretching mode. This confirms the prediction from the sequence alignment that histidine is the proximal ligand. The same spectra were obtained for the B10 Tyr  Leu and Phe mutants (data not shown). The frequency of the FeHis stretching mode is significantly higher than that in any other globin (Table I) and indicates that, unlike other cooperative hemoglobins, there is no proximal strain in this hemoglobin.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   The high frequency resonance Raman spectra of the wild type HbN. A, the ferrous protein at pH 7.5; B, the ferric protein at pH 7.5; C, the ferric protein at pH 10.5. The peaks indicated with × are laser plasma lines.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   The low frequency resonance Raman spectra of the wild type HbN. A, the ferrous protein at pH 7.5; B, the ferric protein at pH 7.5; C, the ferric protein at pH 10.5. Traces D and E correspond to the difference spectrum between H216O and H218O at pH 10.5 for the wild type ferric protein and the B10 Tyr  Phe mutant, respectively.

View this table: [in this window] [in a new window]   Table I The frequencies of the iron-histidine (Fe-His), iron-hydroxide (Fe-OH), iron-CO (Fe-CO) and iron-O2 (Fe-O2) stretching modes of various heme-containing proteins determined by resonance Raman scattering. HS, LS, and 5C stand for high spin, low spin, and five-coordinated species, respectively; ND, not determined.

Ferric HbN-- At neutral pH, the resonance Raman spectrum of the ferric protein is primarily six-coordinate and high spin as indicated by the 1479 and 1561 cm¹ lines for ₃ and ₂, respectively (Fig. 1B). A contribution from a six-coordinate low spin form developed as the pH was raised from 7.5 to 10.5 as indicated by an increase in intensity of the lines at 1501 and 1579 cm¹ for ₃ and ₂, respectively (Fig. 1C). This is consistent with the optical absorption spectra, in which the intensities of bands at 410, 543, and 578 nm increase with respect to those at 406, 503, and 624 nm as the pH is increased. With H₂¹⁶OH₂¹⁸O isotopic substitution at pH 10.5, two isotope sensitive lines were detected at 454 and 561 cm¹ in H₂¹⁶O, which shift to 423 and 533 cm¹, respectively, in H₂¹⁸O (Fig. 2D). Based on the isotope shifts, both of these lines are assigned as FeOH stretching modes. The resonance Raman spectra of the B10 Tyr  Leu and Phe mutants of the ferric protein were also measured. In the B10 Tyr  Phe mutant, the line at 454 cm¹ totally disappeared leaving only the high frequency line, which appeared at about 552 cm¹ in H₂¹⁶O and shifted to 520 cm¹ in H₂¹⁸O (Fig. 2E). Associated with this change in the Fe-OH stretching mode, in the high frequency region of the spectrum, the high spin marker line at 1479 cm¹ is diminished in comparison with the low spin marker line at 1501 cm¹ (data not shown). The Raman spectrum of the B10 Tyr  Leu mutant is not detectable because of a high fluorescence background in this preparation (data not shown).

O₂-bound Ferrous HbN-- In the resonance Raman spectrum of the wild type ferrous oxy-derivative, one line was detected at 560 cm¹ that had oxygen isotope sensitivity (Fig. 3, A and C). In the difference spectrum, a positive peak at 563 cm¹ for ¹⁶O₂ is shifted to 542 cm¹ for ¹⁸O₂, in accord with the predicted shift for a FeO₂ diatomic oscillator of 23 cm¹. As may be seen by inspection of Table I, this mode is lower in HbN than in any other hemoglobin. In the B10 Tyr  Leu mutant, the FeO₂ stretching mode is located at 570 cm¹ for ¹⁶O₂ and shifted to 545 cm¹ with ¹⁸O₂ (Fig. 3, B and D). In the mutant, the FeO₂ stretching mode has the same frequency as that observed in most other hemoglobins and myoglobins.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   The low frequency resonance Raman spectra of the ferrous O2-bound forms of HbN. A, wild type protein; B, the B10 Tyr  Leu mutant at pH 7.5. Traces C and D correspond to the difference spectra between 16O2 and 18O2 for the wild type protein and the B10 Tyr  Leu mutant, respectively.

CO-bound Ferrous HbN-- Unlike the oxy-derivative in which only a single FeO₂ stretching mode was present, two FeCO stretching modes at 534 and 500 cm¹ were detected in the spectrum of the ¹²C¹⁶O derivative of the ferrous protein (Fig. 4A) that shifted to 518 and 483 cm¹ in the ¹³C¹⁸O derivative (Fig. 4C). The relative intensity of these two modes is independent of pH, concentration of the protein, and incident laser power. In the resonance Raman spectra of the B10 Tyr  Leu and Phe mutants, only one CO isotope sensitive line remained in the spectrum which was present at 500 cm¹ for the ¹²C¹⁶O derivative and at 489 cm¹ for the ¹³C¹⁸O derivative (Fig. 4, B and D).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   The low frequency resonance Raman spectra of the ferrous CO-bound forms of HbN. A, the wild type protein; B, the B10 Tyr  Leu mutant at pH 7.5. Traces C and D correspond to the difference spectra between 12C16O and 13C18O for the wild type protein and the B10 Tyr  Leu mutant, respectively. In trace D, the signal at ~1,000 cm1 is assigned as the overtone of the FeCO stretching mode.

In the CO stretching region of the wild type spectrum, CO-isotope sensitive lines were also detected (Fig. 5). A strong line at 1916 cm¹ in ¹²C¹⁶O shifted to 1829 cm¹ in ¹³C¹⁸O. Two lines centered at about 1960 cm¹ in ¹²C¹⁶O derivative appeared as only a single line at 1870 cm¹ in the ¹³C¹⁸O derivative. We assign the lines centered at 1960 cm¹ as originating from a Fermi resonance coupled pair involving a CO stretching mode and a porphyrin mode. Upon the isotope substitution, this coupling is lost so only a single line appears in the ¹³C¹⁸O derivative. Based on these results, we assign the two FeCO stretching modes at 535 and 500 cm¹ as being associated with CO stretching frequencies of 1916 and 1960 cm¹, respectively.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   The high frequency resonance Raman spectra of the ferrous CO-bound forms of HbN at pH 7.5. Traces A and B correspond to 12C16O and 13C18O, respectively. Trace C is the difference spectrum between traces A and B. Trace D, the correlation diagram of the FeCO stretching frequency and the CO stretching frequency for a variety of heme proteins and porphyrin derivatives. The points for HbN (the filled circles) lie on the imidazole/histidine correlation line.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tyrosine Hydrogen Bonding-- The frequencies of the various iron-ligand stretching modes of HbN reported here are compared with those of several other heme-containing proteins listed in Table I. It is evident from this table that the distal heme pocket of HbN is unique among the globins. When the B10 Tyr is mutated to either Leu or Phe, the iron-exogenous ligand modes convert to values similar to those of other globins, indicating that the B10 Tyr plays an important role in modulating the ligand binding properties of HbN. On the other hand, the FeHis stretching frequency of HbN is not affected by the mutation at the B10 position, indicating that the changes in the distal environment do not propagate to the proximal side of the heme.

In vertebrate globins, the bound oxygen is stabilized by the distal histidine at the E7 position through hydrogen bonding to the terminal oxygen atom of O₂ as illustrated in Fig. 6 (13-15). The E7 residue in most invertebrate globins, on the other hand, is occupied by a glutamine (1). Stabilization of the ligand through the distal (E7) glutamine has been observed in Lucina pectinata and Ascaris suum hemoglobins (16). Intriguingly, the E7 Gln does not seem to play a crucial role in stabilizing the bound ligands in bacterial hemoglobins, apparently because of conformational constraints on this residue imposed by the polypeptide architecture. For example, the E7-10 region of the polypeptide in Vitreoscilla, instead of adopting the typical -helical conformation, is disordered (17). As a result, the E7 Gln is rotated out of the heme pocket. Spectral and kinetic studies of the binding of oxygen and CO to E7 mutants of Vitreoscilla hemoglobin showed that this substitution had little effect on the ligand binding properties of this protein (18), evidence that E7 Gln does not hydrogen-bond to the bound ligand. The same segment in another bacterium, Alcaligenes eutrophus, adopts an elongated and well defined E-helix but deviates significantly from that in other globins by more than 10° because of an increase in the angle between E and F helices (19). This leads to a much more open distal pocket and causes a much larger entropic energy barrier for the E7 residue to interact with heme bound ligands.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   The hydrogen bonding network for stabilizing the bound ligands in myoglobin (Mb), A. suum Hb, and HbN.

Ligand stabilization in invertebrate and bacterial hemoglobins can also be achieved through a distal tyrosine at the B10 position (1, 17). When the hydroxyl group of the B10 Tyr is properly positioned in the heme distal site as in the Hb from Ascaris (Fig. 6), very low ligand dissociation rates are observed (20). In contrast, the oxygen dissociation rate of a myoglobin mutant with the B10 Leu mutated to Tyr is increased because of the unfavorable B10 Tyr-O₂ contacts (20, 21).

In HbN, the E7 position is occupied by Leu. Thus, possible stabilization of heme-bound ligands by this residue is excluded. On the other hand, the role of the B10 residue in HbN on the stabilization of the bound oxygen is clear. The frequency of the FeO₂ stretching mode of HbN (560 cm¹) is very low as compared with that in other globins in which the mode is located at approximately 570 cm¹ (22). In general, as the OO bond of the bound O₂ is highly polarized by the heme iron, the FeO₂ stretching frequency is relatively insensitive to distal residue mutations (22), but in HbN the frequency of the FeO₂ mode is very sensitive to the distal B10 mutation. We propose that the low frequency of the FeO₂ stretching mode in wild type HbN is the result of a unique hydrogen bonding pattern between the bound oxygen and the distal Tyr, as illustrated in Fig. 6, in which the bound oxygen is hydrogen-bonded to the distal Tyr through the sp² orbital of the proximal oxygen atom. This hydrogen bonding directly constrains and weakens the FeO₂ bond as indicated by the low FeO₂ stretching frequency. Mutation of the B10 Tyr to Leu or Phe releases the oxygen, and thus the frequency of the FeO₂ stretching mode shifts to 570 cm¹, a frequency identical to that in the vertebrate globins.

A similar type of hydrogen bond has been found in A. suum Hb between a distal Gln and the bound oxygen based on crystallographic studies (23) as illustrated in Fig. 6, in which the bound oxygen is hydrogen-bonded to a distal glutamine and a tyrosine through the proximal and terminal oxygen atoms, respectively. Like HbN, two conformations of the CO-bound form were observed in Ascaris Hb. The FeCO stretching mode (543 cm¹) in the closed conformation is higher than that of HbN. It has been postulated that the high frequency is a consequence of hydrogen bonding to the CO by both the Gln and the Tyr residues. Mutating B10 Tyr to Phe in Ascaris Hb causes the frequency to shift down to 520 cm¹, reflecting the loss of the hydrogen bonding contribution from the Tyr (50).

In the CO-derivative of HbN, the B10 tyrosine mutation causes the disappearance of the line at 535 cm¹, with only the 500 cm¹ line remaining. Therefore, we assign the high frequency mode located at 535 cm¹ as originating from a closed conformation in which the distal B10 Tyr strongly interacts with the CO as illustrated in Fig. 6 (conformational rearrangements may be required for the B10 Tyr to interact the oxygen atom of the CO). This is a positive polar interaction which we attribute to hydrogen bonding with the proton on the tyrosine. It is well established that positive polar interactions weaken the CO bond (lowering its stretching frequency) and strengthening the FeCO bond (increasing its stretching frequency) (24-26). The 500 cm¹ band is assigned as an open conformation without this strong hydrogen bonding interaction. This latter frequency is similar to that reported in vertebrate globins.

Because of the electronic structure of the FeCO moiety, when the distal interactions strengthen the CO bond and concomitantly weaken the FeCO bond (or vice versa), there is an inverse correlation curve relating the frequencies of the FeCO stretching modes with those of the associated CO stretching modes (27-29). These curves depend on the identity of the proximal ligand, such that the curve for imidazole/histidine is distinct from that of thiolate. Both of the CO-bound structures of HbN fall on the imidazole/histidine correlation curve as shown in Fig. 5D. This is consistent with the assignment of the proximal ligand as a histidine and the frequency difference between the two forms resulting from a hydrogen-bonding interaction between a distal residue and the CO. Furthermore, because the two data points fall on the same correlation curve, the presence of this hydrogen bond does not affect the HisFeCO -bonding system.

The presence of two conformations in the carbon monoxide derivative, in contrast to a single conformation for the oxy-derivative, is attributed to the difference in the intrinsic hydrogen bonding strength between tyrosine and the bound ligand as well as their relative orientation. The bound O₂ in the oxy-derivative may be optimally positioned for forming the hydrogen bond with the B10 Tyr as illustrated in Fig. 6. It is locked in the closed conformation because the tyrosine forms a very strong hydrogen bond with the O₂ such that the gain in enthalpy overcomes the loss in the conformational entropy. In contrast, the intrinsic hydrogen bonding strength between CO and Tyr is weaker, and the conformational entropy cost is high because of a preferred linear geometry of the bound CO and the location of the B10 Tyr in HbN which is optimized for forming a hydrogen bond with the proximal oxygen atom of the bound O₂ (Fig. 5). As a result, the gain in enthalpy is not enough to compensate for the loss in conformational entropy such that the carbon-monoxide derivative is in a thermal equilibrium between an open and a closed conformation.

Feis et al. (30) carried out an in-depth study of hydroxide modes in heme proteins. The FeOH stretching mode is found at 550 and 553 cm¹ in low spin metmyoglobin and methemoglobin, respectively, and at 491 and 492 cm¹ for the high spin forms of these proteins (Table I). The two hydroxy modes of HbN were identified at 456 and 560 cm¹ for the high and low spin forms, respectively. The observation that the FeOH stretching mode of the high spin species appears nearly 40 cm¹ lower than in other hemoglobins or myoglobins confirms the presence of the very strong hydrogen bond between the hydroxide moiety and the B10 Tyr. A similar shift is detected in the low spin form of horseradish peroxidase (30), in which the mode is detected at 503 cm¹, 50 cm¹ lower than the low spin forms of the globins. This low frequency has also been attributed to very strong hydrogen bonding to the hydroxide in HRP.

The Physiological Function of HbN-- In a previous report, we postulated that the physiological function of HbN is to protect the M. tuberculosis bacterium against attack by NO during its latent phase (10). Similar functions have been proposed for newly discovered flavohemoglobins in bacteria (7-9), suggesting that the detoxification of NO may be a more ancient function for the widely distributed hemoglobins (31). The structural features of HbN revealed by resonance Raman scattering are distinct from those of other globins. These properties demonstrate that the iron-bound ligands strongly interact with the tyrosine in the distal pocket. Such interactions are absent in normal oxygen storage and transport globins but are present in other heme-containing proteins that are involved in oxygen chemistry such as the peroxidases (32). These data thereby suggest that the unique heme pocket of HbN may be optimized for performing oxygen chemistry with NO.

It was demonstrated in vertebrate hemoglobin that the oxy-derivative reacts with NO and results in nitrate (33, 34). Recent kinetic studies suggest that this reaction goes through a peroxynitrite intermediate (Hb⁺³OONO) as described in Equation 1.

Because the conversion from the peroxynitrite intermediate to nitrate and a ferric protein must involve OO bond cleavage, the reaction is proposed to proceed through an oxy-ferryl (Fe⁺⁴=O) intermediate as observed in the reaction between NO and model porphyrin compounds (35) and illustrated in Equation 2 or Equation 3.

(Eq. 1)

(Eq. 2)

(Eq. 3)

In Equations 2 and 3, the reaction goes through a compound I, a heterolytic OO bond cleavage product, and compound II, a homolytic OO bond cleavage product, respectively (36, 37). Both the compound I and II species have been identified in the catalytic cycle of peroxidases. To generate compound I-like species, the iron needs to receive one electron from either the porphyrin or a nearby amino acid residue which is indicated as R^+· in Equation 2. A good candidate for this amino acid residue in HbN is the B10 Tyr which may supply the redox equivalent needed for catalyzing the OO bond heterolytic cleavage. More kinetic studies are required to confirm this model although the oxy-ferryl species may not be observable if the OO bond breakage is much slower than the subsequent reaction to NO₃.

Implications for Structure of the Binuclear Site in Cytochrome c Oxidase-- The only other heme protein that has a frequency for its FeOH stretching mode as low as that reported here for HbN (454 cm¹) is cytochrome c oxidase, in which the mode is detected at 450 cm¹. No suitable explanation for the origin of this low frequency has been put forth. Cytochrome c oxidase catalyzes the reduction of oxygen to water through a complex series of intermediates. Recent crystallographic data have shown that one of the Cu_B ligands, His-240, is cross-linked to Tyr-244 at the heme a₃-Cu_B catalytic site of the enzyme and that this cross-linked tyrosine is ideally positioned to participate in the dioxygen reduction (38). The experiments reported here give support to the proposal that the tyrosine in cytochrome c oxidase plays a critical role in interacting with the bound ligands. The similarity of the stretching frequencies of the FeOH stretching modes in these two proteins, which are significantly different from those in any other heme protein that have been reported, suggests that the two proteins have similar bonding motifs for the hydroxide derivatives. To test if this tyrosine modifies the functional and the spectroscopic properties of cytochrome c oxidase, site-directed mutagenesis studies have been reported. Unfortunately, the tyrosine plays such a critical role in stabilizing the structure of the protein that the mutants were nonfunctional and the catalytic site was completely disrupted (39). Thus, no conclusions could be drawn regarding the role of this residue from the mutagenesis experiments.

In some of the intermediates in cytochrome c oxidase, oxy-ferryl structures are formed. It has been proposed that a reducing equivalent for the formation of this intermediate can reside on the Tyr-244 (40). Support for this idea has come from EPR measurements in which a radical signal from a tyrosine residue was detected (41). Thus, it is quite possible that this radical is an intermediate in the mechanism of oxygen reduction. The putative tyrosine radical is thought to be re-reduced by the subsequent electron transfer events. A similar functional role for the B10 tyrosine of HbN is proposed here during the conversion of NO to nitrate as illustrated in Equation 2. In this case, the tyrosine radical intermediate is presumably re-reduced by the substrate, NO₂. It is interesting to consider that these very different proteins may exploit very similar mechanisms to carry out their reaction chemistry.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: (718) 430-4234; Fax: (718) 430-4230; E-mail: syeh@aecom.yu.edu.

	ABBREVIATIONS

The abbreviations used are: HbN, homodimeric hemoglobin; CCP, cytochrome c peroxidase; HRP, horse radish peroxidase; CCO, cytochrome c oxidase; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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