A change in the heme stereochemistry of cytochrome c upon addition of sodium dodecyl sulfate: electron paramagnetic resonance and electronic absorption spectral study

Electron paramagnetic resonance and electronic absorption spectral changes upon addition of sodium dodecyl sulfate (SDS) to ferric and ferrous cytochrome c have been measured at 77 degrees K and at room temperature. The spectral changes upon addition of SDS to ferric cytochrome c were performed, in two steps, from native low-spin to another low-spin spectrum and subsequently to high-spin-like spectrum. On the other hand, the spectral changes upon addition of SDS to ferrous cytochrome c proceeded, in one step, from native low-spin to high-spin spectrum. The high-spin-like spectrum of ferric cytochrome c and the high-spin spectrum of ferrous cytochrome c in the presence of high concentrations of SDS are, respectively, apparently similar to those of ferric and ferrous cytochrome c' at physiological pH in spectral features. These spectral similarities suggest the similarities in the heme stereochemistry and the ground state of heme iron. Further, the spectra of cytochrome c in the presence of SDS varied with the change of pH values. The ferric high-spin-like and ferrous high-spin spectra were stable at neutral pH and below it. Conformational changes of cytochrome c upon addition of SDS are also discussed.     

Paramagnetic properties of the low- and high-spin states of yeast cytochrome c peroxidase

Here we describe paramagnetic NMR analysis of the low- and high-spin forms of yeast cytochrome c peroxidase (CcP), a 34 kDa heme enzyme involved in hydroperoxide reduction in mitochondria. Starting from the assigned NMR spectra of a low-spin CN-bound CcP and using a strategy based on paramagnetic pseudocontact shifts, we have obtained backbone resonance assignments for the diamagnetic, iron-free protein and the high-spin, resting-state enzyme. The derived chemical shifts were further used to determine low- and high-spin magnetic susceptibility tensors and the zero-field splitting constant (D) for the high-spin CcP. The D value indicates that the latter contains a hexacoordinate heme species with a weak field ligand, such as water, in the axial position. Being one of the very few high-spin heme proteins analyzed in this fashion, the resting state CcP expands our knowledge of the heme coordination chemistry in biological systems.     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

Proton NMR study of the heme environment in bacterial quinol oxidases

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.     

NMR and electron-paramagnetic-resonance studies of a dihaem cytochrome from Pseudomonas stutzeri (ATCC 11607) (cytochrome c peroxidase)

A dihaem cytochrome (Mr 37 400) with cytochrome c peroxidase activity was purified from Pseudomonas stutzeri (ATCC 11 607). The haem redox potentials are far apart: one of the haems is completely ascorbate-reducible and the other is only reduced by dithionite. The coordination, spin states and redox properties of the covalently bound haems were probed by visible, NMR and electron paramagnetic resonance (EPR) spectroscopies in three oxidation states. In the oxidized state, the low-temperature EPR spectrum of the native enzyme is a complex superimposition of three components: (I) a low-spin haem indicating a histidinyl-methionyl coordination; (II) a low-spin haem indicating a histidinyl-histidinyl coordination; and (III) a minor high-spin haem component. At room temperature, NMR and optical studies indicate the presence of high-spin and low-spin haems, suggesting that for one of the haems a high-spin to low-spin transition is observed when temperature is decreased. In the half-reduced state, the component I (high redox potential) of the EPR spectrum disappears and induces a change in the g-values and linewidth of component II; the high-spin component II is no longer detected at low temperature. Visible and NMR studies reveal the presence of a high-spin ferric and a low-spin (methionyl-coordinated) ferrous state. The NMR data fully support the haem-haem interaction probed by EPR. In the reduced state, the NMR spectrum indicates that the low-potential haem is high-spin ferrous.     

The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c.

The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.     

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.     

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH.

The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70. This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra. Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies. The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH. Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90. Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH. However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.     

Low temperature magnetic susceptibility of a human Co(II) carbonic anhydrase B sulphonamide complex.

The magnetic susceptibility of the acetazolamide complex of human Co(II) carbonic anhydrase B was measured between 1.4 and 160 K. From the temperature dependence of the paramagnetic contribution of Co(II) a g-value of 2.23 +/- 0.02 and a zero-field splitting of (33 +/- 1) cm-1 were estimated. The effective number of Bohr magnetons as T leads to 0 is 3.35 in excellent agreement with the number (3.38) calculated from the apparent g'-values of the EPR spectrum at 16 K. Extrapolation to high temperatures gave an effective number of Bohr magnetons of 4.32.     

A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Spectroscopic studies of partially reduced forms of Wolinella succinogenes nitrite reductase

Reductive titrations of the dissimilatory hexa-haem nitrite reductase, Wolinella succinogenes, with methyl viologen semiquinone (MV) and sodium dithionite, have been followed at room temperature by absorption, natural (CD) and magnetic circular dichroism (MCD) spectroscopies and at liquid helium temperature by electron paramagnetic resonance (EPR) and MCD spectroscopies. The nature of the reduced enzyme depends on the reductant employed. At room temperature a single high-spin ferrous haem, observed by MCD after reduction with MV, is absent from dithionite reduced samples. It is suggested that a product of dithionite oxidation becomes bound with high affinity to the reduced state of the enzyme causing the ferrous haem to become low-spin. The site occupied is likely to be the substrate binding haem. The course of the titration with MV at room temperature shows the reduction of high-spin ferric to high-spin ferrous haem. Since the EPR spectrum reveals the presence of an unusual high-low spin ferric haem pair in the oxidised state we propose that the active site of the enzyme is a novel haem pair consisting of one high (5-coordinate) and one low-spin (6 coordinate) haem, magnetically coupled and possibly bridged by a histidinate ligand.     

Structural and functional insights of GSU0105, a unique multiheme cytochrome from G. sulfurreducens

Geobacter sulfurreducens possesses over 100 cytochromes that assure an effective electron transfer to the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA family, composed of five periplasmic triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome synthetized by G. sulfurreducens in Fe(III)-reducing conditions but is not present in cultures grown on fumarate. This cytochrome has a low sequence identity with the PpcA family cytochromes and a different heme coordination, based on the analysis of its amino acid sequence. In this work, amino acid sequence analysis, site-directed mutagenesis, and complementary biophysical techniques, including ultraviolet-visible, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopies, were used to characterize GSU0105. The cytochrome has a low percentage of secondary structural elements, with features of α-helices and β-sheets. Nuclear magnetic resonance shows that the protein contains three low-spin hemes (Fe(II), S = 0) in the reduced state. Electron paramagnetic resonance shows that, in the oxidized state, one of the hemes becomes high-spin (Fe(III), S = 5/2), whereas the two others remain low-spin (Fe(III), S = 1/2). The data obtained also indicate that the heme groups have distinct axial coordination. The apparent midpoint reduction potential of GSU0105 (−154 mV) is pH independent in the physiological range. However, the pH modulates the reduction potential of the heme that undergoes the low- to high-spin interconversion. The reduction potential values of cytochrome GSU0105 are more distinct compared to those of the PpcA family members, providing the protein with a larger functional working redox potential range. Overall, the results obtained, together with an amino acid sequence analysis of different multiheme cytochrome families, indicate that GSU0105 is a member of a new group of triheme cytochromes.     

The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.

The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide.     

Assignment of heme methyl 1H-NMR resonances of high-spin and low-spin ferric complexes of cytochrome p450cam using one-dimensional and two-dimensional paramagnetic signals enhancement (PASE) magnetization transfer experiments.

An 1H-NMR study of ferric cytochrome P450cam in different paramagnetic states was performed. Assignment of three heme methyl resonances of the isocyanide adduct of cytochrome P450 in the ferric low-spin state was recently performed using electron exchange in the presence of putidaredoxin [Mouro, C., Bondon, A., Jung, C., Hui Bon Hoa, G., De Certaines, J.D., Spencer, R.G.S. & Simonneaux, G. (1999) FEBS Lett. 455, 302-306]. In this study, heme methyl protons of cytochrome P450 in the native high-spin and low-spin states were assigned through one-dimensional and two-dimensional magnetization transfer spectroscopy using the paramagnetic signals enhancement (PASE) method. The order of the methyl proton chemical shifts is inverted between high-spin and low-spin states. The methyl order observed in the ferric low-spin isocyanide complexes is related to the orientation of the cysteinate ligand.     

Some magnetic properties of Pseudomonas cytochrome oxidase.

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.     

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Sch?fer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.