Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Evaluating the roles of the heme a side chains in cytochrome c oxidase using designed heme proteins

Heme a is a redox cofactor unique to cytochrome c oxidases and vital to aerobic respiration. Heme a differs from the more common heme b by two chemical modifications, the C-8 formyl group and the C-2 hydroxyethylfarnesyl group. The effects of these porphyrin substituents on ferric and ferrous heme binding and electrochemistry were evaluated in a designed heme protein maquette. The maquette scaffold chosen, [Delta7-H3m](2), is a four-alpha-helix bundle that contains two bis(3-methyl-l-histidine) heme binding sites with known absolute ferric and ferrous heme b affinities. Hemes b, o, o+16, and heme a, those involved in the biosynthesis of heme a, were incorporated into the bis(3-methyl-l-histidine) heme binding sites in [Delta7-H3m](2). Spectroscopic analyses indicate that 2 equiv of each heme binds to [Delta7-H3m](2), as designed. Equilibrium binding studies of the hemes with the maquette demonstrate the tight affinity for hemes containing the C-2 hydroxyethylfarnesyl group in both the ferric and ferrous forms. Coupled with the measured equilibrium midpoint potentials, the data indicate that the hydroxyethylfarnesyl group stabilizes the binding of both ferrous and ferric heme by at least 6.3 kcal/mol via hydrophobic interactions. The data also demonstrate that the incorporation of the C-8 formyl substituent in heme a results in a 179 mV, or 4.1 kcal/mol, positive shift in the heme reduction potential relative to heme o due to the destabilization of ferric heme binding relative to ferrous heme binding. The two substituents appear to counterbalance each other to provide for tighter heme a affinity relative to heme b in both the ferrous and ferric forms by at least 6.3 and 2.1 kcal/mol, respectively. These results also provide a rationale for the reaction sequence observed in the biosynthesis of heme a.     

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.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.     

Effect of ligands of ferric hemes on interaction between ferric and ferrous chains in partially oxidized hemoglobin A.

Normal values of Bohr effect of oxygenation of partially oxidized hemoglobin A with ferrihemes liganded either with H2O and OH or with CN have been found in the range of pH values from 6.8 to 7.6 in 45 micrometer (Fe)-hemoglobin containing 36--38% of ferrihemes. As the changes of oxygen affinity of Hb A induced by changes of pH are due to the modifications of R state, this quaternary conformation is thought to be unchanged in the studied of R state, this quaternary conformation is thought to be unchanged in the studied forms of partially oxidized hemoglobin. It is suggested that interactions between ferric and ferrous hemes leading to the increased affinity of ferrous hemes to oxygen occur in deoxygenated form of partially oxidized hemoglobin. In partially oxidized hemoglobin with ferric hemes liganded with H2O asymmetry of oxygen binding curves has been noted, which is not observed in forms with ferric hemes liganded with OH ot CN. This shows the effect of ligands of ferric hemes on interactions between chains containing ferric and ferrous hemes.     

Nitrite reductase activity of hemoglobin S (sickle) provides insight into contributions of heme redox potential versus ligand affinity

Hemoglobin A (HbA) is an allosterically regulated nitrite reductase that reduces nitrite to NO under physiological hypoxia. The efficiency of this reaction is modulated by two intrinsic and opposing properties: availability of unliganded ferrous hemes and R-state character of the hemoglobin tetramer. Nitrite is reduced by deoxygenated ferrous hemes, such that heme deoxygenation increases the rate of NO generation. However, heme reactivity with nitrite, represented by its bimolecular rate constant, is greatest when the tetramer is in the R quaternary state. The mechanism underlying the higher reactivity of R-state hemes remains elusive. It can be due to the lower heme redox potential of R-state ferrous hemes or could reflect the high ligand affinity geometry of R-state tetramers that facilitates nitrite binding. We evaluated the nitrite reductase activity of unpolymerized sickle hemoglobin (HbS), whose oxygen affinity and cooperativity profile are equal to those of HbA, but whose heme iron has a lower redox potential. We now report that HbS exhibits allosteric nitrite reductase activity with competing proton and redox Bohr effects. In addition, we found that solution phase HbS reduces nitrite to NO significantly faster than HbA, supporting the thesis that heme electronics (i.e. redox potential) contributes to the high reactivity of R-state deoxy-hemes with nitrite. From a pathophysiological standpoint, under conditions where HbS polymers form, the rate of nitrite reduction is reduced compared with HbA and solution-phase HbS, indicating that HbS polymers reduce nitrite more slowly.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modeling low-pH hemoproteins

A tetracoordinate ferrous heme (iron-porphyrin) has been proposed as an intermediate at low pH (less than 3.0) for respiratory hemoproteins, peroxidases, and model heme complexes. This intermediate is believed to arise via protonation of the N(epsilon) atom of the proximal histidine and consequent cleavage of the Fe-N(epsilon) bond. To establish a spectral signature for the proposed low-pH tetracoordinate species, we have obtained Soret excitation resonance Raman spectra on samples of crystallographically defined, tetracoordinate iron(II)-octaethylporphyrin (Fe.OEP; S = 1). The high-frequency (greater than or equal to 900 cm-1) resonance Raman spectral features of Fe.OEP are clearly distinct from those of high-spin pentacoordinate or low-spin hexacoordinate ferrous hemes. Rather, they are at frequencies more typically observed for low-spin hexacoordinate ferric porphyrins. Comparative spectral analysis of tetracoordinate Fe.OEP and other proposed tetracoordinate ferrous hemes (e.g. iron(II)-protoporphyrin IX) demonstrates little or no macrocycle effect on the resonance Raman frequencies above 900 cm-1. This work thus serves to provide a testable spectral signature by which the existence of the proposed tetracoordinate biological intermediate may be verified and by which its functional significance may be tested.     

Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR.

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em = +280 mV, 30 degrees; gz = 3.33, Em = 0 mV, 45 degrees; gz = 3.03, Em = +95 mV, 40-50 degrees and gz = 2.95, Em = +150 mV, 90 degrees. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the +95 mV and the +150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.     

Thermodynamic redox behavior of the heme centers of cbb3 heme-copper oxygen reductase from Bradyrhizobium japonicum

A comprehensive study of the thermodynamic redox behavior of the hemes from the cbb3 oxygen reductase from Bradyrhizobium japonicum was performed. This enzyme is a member of the C-type heme-copper oxygen reductase superfamily and has three subunits with six redox centers: four low-spin hemes and a high-spin heme and one copper ion, composing the site where oxygen is reduced. In this analysis, the visible spectra and redox properties of the five heme centers were deconvoluted. Their redox profiles and the pH dependence of the midpoint reduction potentials (redox-Bohr effect) were investigated. The reference reduction potentials (defined for a state where all centers are reduced) and homotropic interaction potentials were determined in the framework of a model of pairwise interacting redox centers. At pH 7.7, the reference reduction potentials for the three hemes c are 390, 300, and 220 mV, with low interaction potentials between them, weaker than -15 mV. For hemes b and b3, reference reduction potentials of 375 and 290 mV, respectively, were obtained; these two redox centers show an interaction potential weaker than -60 mV. The midpoint reduction potentials of all five hemes are pH-dependent. The study of these thermodynamic parameters is important in understanding the coupling mechanism of the redox and chemical processes during oxygen reduction. The analysis of the thermodynamic redox behavior of the cbb3 oxygen reductase contributes to the investigation of the mechanism of electron transfer and proton translocation by heme-copper oxygen reductases in general and indicates a thermodynamic coupling for the electron and proton transfer mechanisms.     

Influence of globin structures on the state of the heme. Ferrous low spin derivatives.

Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.     

Tuning heme redox potentials in the cytochrome C subunit of photosynthetic reaction centers

The photosynthetic reaction center (RC) from Rhodopseudomonas viridis contains four cytochrome c hemes. They establish the initial part of the electron transfer (ET) chain through the RC. Despite their chemical identity, their midpoint potentials cover an interval of 440 mV. The individual heme midpoint potentials determine the ET kinetics and are therefore tuned by specific interactions with the protein environment. Here, we use an electrostatic approach based on the solution of the linearized Poisson-Boltzmann equation to evaluate the determinants of individual heme redox potentials. Our calculated redox potentials agree within 25 meV with the experimentally measured values. The heme redox potentials are mainly governed by solvent accessibility of the hemes and propionic acids, by neutralization of the negative charges at the propionates through either protonation or formation of salt bridges, by interactions with other hemes, and to a lesser extent, with other titratable protein side chains. In contrast to earlier computations on this system, we used quantum chemically derived atomic charges, considered an equilibrium-distributed protonation pattern, and accounted for interdependencies of site-site interactions. We provide values for the working potentials of all hemes as a function of the solution redox potential, which are crucial for calculations of ET rates. We identify residues whose site-directed mutation might significantly influence ET processes in the cytochrome c part of the RC. Redox potentials measured on a previously generated mutant could be reproduced by calculations based on a model structure of the mutant generated from the wild type RC.     

The Photo-Oxidation of Low-Potential Hemes in the Tetraheme Cytochrome Subunit of the Reaction Center in Whole Cells of Blastochloris viridis

The reduction and the photo-oxidation of the low-potential hemesin the tetraheme cytochrome subunit of the photosynthetic reactioncenter complex (RC) of the purple non-sulfur bacterium Blastochloris(Rhodopseudomonas) viridis was observed in whole cells afterlong incubation in the dark. The low-potential hemes showedfast photo-oxidation, similar to the high-potential hemes, asthe common feature of the hemes in the RC-bound tetraheme cytochrome;while their re-reduction was very slow, compared to those ofthe high-potential hemes. Myxothiazol, a specific inhibitorof the cytochrome bc₁ complex, had only minor effect on there-reduction of low-potential hemes, suggesting that low-potentialhemes are not efficiently re-reduced by the electrons from thecytochrome be, complex and ubiquinones. These results showedthe participation of low-potential hemes in photosynthetic electrontransfer in vivo. The physiological function of the low-potentialhemes in vivo is discussed. (Received July 23, 1998; Accepted November 30, 1998)     

A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.     

Kinetic study of the reduction mechanism for Desulfovibrio gigas cytochrome c3.

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish-Bowden, A. & Koshland, D.E. Jr (1970) J. Biol. Chem. 245, 6241-6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J.J. & Xavier, A.V. (1991) Eur. J. Biochem. 202, 1101-1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network.     

Interaction of cytochrome c, ferrous ion, and phosphate. Electron transfer within a stoichiometric complex.

The rate and extent of electron transfer from ferrous ion to ferricytochrome c are enhanced by the presence of inorganic orthophosphate at concentrations comparable to those of reductant and oxidant. Evidence, obtained by the method of continuous variations, shows that the electron transfer occurs within a stoichiometric complex composed of cytochrome c, ferrous ion, and phosphate in molar proportions of about 1:1:1. The incorporation of the anion into this complex appears to result in a modulation of the extent and rate of cytochrome c reduction. The rate of electron transfer obeys a first order rate law, characterized by an apparent first order rate constant of 1.4 min-1. The complex has kinetic significance only; equilibrium dialysis, gel filtration, and sedimentation velocity experiments yielded no evidence for stable binding of phosphate and iron, or of aggregation, on a significant scale. The extent of reduction is limited (for reasons not yet known) to about one-half of the available cytochrome molecules. Reduction in excess of 50% can be achieved only when both, ferrous ion and phosphate, are present in excess of the cytochrome concentration. Kinetic data indicate that reduction to extents over and below 50% occurs by different mechanisms.