Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies.

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).     

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.     

Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes.

Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-558 (cyt b-558). A thick cell suspension (approximately 2 x 10(9) cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O2(-)-forming NADPH oxidase associated in the membranes was solubilized with heptyl-thio-glucoside at 0 degree C and concentrated up to 45 microM cyt b-558 with no modification of the heme moiety confirmed by its O2(-)-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 +/- 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.     

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.     

Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

Formation of a bis(histidyl) heme iron complex in manganese peroxidase at high pH and restoration of the native enzyme structure by calcium

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high- to low-spin alkaline transition occurs at approximately 2 pH units lower in an F190I mutant MnP when compared to the wild-type enzyme. Herein, we provide evidence that these spectral changes are attributable to the formation of a bis(histidyl) heme iron complex in both proteins at high pH. The resonance Raman (RR) spectra of both ferric proteins at high pH are similar, indicating similar heme environments in both proteins, and resemble that of ferric cytochrome b(558), a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visible spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 nm, resembling the absorption spectrum of ferrous cytochrome b(558). RR spectra of the reduced wild-type and F190I mutant proteins at high pH are also similar to the RR spectrum of ferrous cytochrome b(558), further suggesting that the alkaline low-spin species is a bis(histidyl) heme derivative. No shift in the low-frequency RR bands was observed in 75% (18)O-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that addition of Ca(2+) to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn(2+), Mg(2+), Zn(2+), or Cd(2+), do not restore the enzyme under the conditions studied.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

The cytochromes of anaerobically grown Escherichia coli. An electron-paramagnetic-resonance study of the cytochrome bd complex in situ.

The e.p.r. signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant. Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d. These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d. At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy). The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH. A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558. The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.     

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.     

Role of calcium in metalloenzymes: effects of calcium removal on the axial ligation geometry and magnetic properties of the catalytic diheme center in MauG

MauG is a diheme enzyme possessing a five-coordinate high-spin heme with an axial His ligand and a six-coordinate low-spin heme with His-Tyr axial ligation. A Ca(2+) ion is linked to the two hemes via hydrogen bond networks, and the enzyme activity depends on its presence. Removal of Ca(2+) altered the electron paramagnetic resonance (EPR) signals of each ferric heme such that the intensity of the high-spin heme was decreased and the low-spin heme was significantly broadened. Addition of Ca(2+) back to the sample restored the original EPR signals and enzyme activity. The molecular basis for this Ca(2+)-dependent behavior was studied by magnetic resonance and M?ssbauer spectroscopy. The results show that in the Ca(2+)-depleted MauG the high-spin heme was converted to a low-spin heme and the original low-spin heme exhibited a change in the relative orientations of its two axial ligands. The properties of these two hemes are each different than those of the heme in native MauG and are now similar to each other. The EPR spectrum of Ca(2+)-free MauG appears to describe one set of low-spin ferric heme signals with a large g(max) and g anisotropy and a greatly altered spin relaxation property. Both EPR and M?ssbauer spectroscopic results show that the two hemes are present as unusual highly rhombic low-spin hemes in Ca(2+)-depleted MauG, with a smaller orientation angle between the two axial ligand planes. These findings provide insight into the correlation of enzyme activity with the orientation of axial heme ligands and describe a role for the calcium ion in maintaining this structural orientation that is required for activity.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

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.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g ～ 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undetectable components reduced appears to be inversely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during the titration. At pH 9.3 the quantity of heme represented in the high-spin signals is < 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

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.     

Oxidation-reduction potentials of the hemes in cytochrome C3 from Desulfovibrio gigas in the presence and absence of ferredoxin by EPR spectroscopy.

1. Ferricytochrome c3 from D. gigas exhibits two low-spin ferric heme EPR resonances with gz-values at 2.959 and 2.853. Ferrocytochrome c3 is diamagnetic based on the absence of any EPR signals. 2. EPR potentiometric titrations result in the resolution of the two low-spin ferric heme resonances into two additional heme components representing in total the four hemes of the cytochrome, with EM values of -235 mV and -315 mV at heme resonance I and EM values of -235 mV and -306 mV at heme resonance II. 3. EPR spectroscopy has detected a significant diminution of intensity (approx. 60 p. 100) in the gx amplitude of ferricytochrome c3 in the presence of D. gigas ferredoxin II. The presence of ferredoxin II also causes a more negative shift in the EM of the second components of the signals at heme resonances I and II of cytochrome C3. Both observations suggest that an interaction has occurred between cytochrome C3 and ferredoxin II. 4. The results presented suggest that the heme ligand environment of ferricytochrome c3 from D. gigas is less perturbed and/or less asymmetric than environment for ferricytochrome c3 from D. vulgaris whose EPR behavior indicates the non-equivalence of all four hemes.     

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.     

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mo?ssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mo?ssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ～ 6 and g ～ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ～ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.