Horse heart ferricytochrome c: conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III state IIIa state II state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Horse heart ferricytochromec: Conformation and heme configuration of high ionic strength acidic forms

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s ?state III _s,a ?state II _s ?state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a →state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration

The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the F^III and F^II CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pK _a values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of F^II, with F^III being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, F^II CT-cytochrome c being the most deranged.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

A possible role for the covalent heme-protein linkage in cytochrome c revealed via comparison of N-acetylmicroperoxidase-8 and a synthetic, monohistidine-coordinated heme peptide

N-Acetylmicroperoxidase-8 (1) contains heme and residues 14-21 of horse mitochondrial cytochrome c (cyt c). The two thioether bonds linking protein to heme in cyt c are present in 1, and the native axial ligand His-18 remains coordinated to iron. As an approach to probing structural or functional roles played by the double covalent heme-protein linkage in cyt c, we have initiated a study in which the properties of 1 are compared with those of a synthetic mono-His coordinated heme peptide containing a single covalent linkage (2). One consequence of the greater conformational restriction imposed on peptide conformation in 1 is that His-Fe(III) coordination is approximately 1.4 kcal/mol more favorable in 1 than in 2. This highlights a clear advantage conferred to cyt c by having two covalent heme-protein linkages rather than one: greater thermodynamic stability of the protein fold. EPR (11 K) and resonance Raman (298 K) studies reveal that 1 and 2 exhibit a thermal high-spin/low-spin ferric equilibrium but that low-spin character is considerably more pronounced in 1. In addition, the thioether 2-(methylthio)ethanol (MTE) coordinates 0.5 kcal/mol more strongly to 1 than to 2 in 60:40 H(2)O/CH(3)OH and only triggers the expected conversion of iron to the low-spin state characteristic of ferric cyt c in the case of 1. This demonstrates that the axial ligand field provided by an imidazole and a thioether is too weak to induce a high-spin to low-spin conversion in a ferric porphyrin. Our results suggest that a conformationally constrained double covalent heme-protein linkage, as exists in 1 and its parent protein cyt c, is an effective solution that nature has evolved to circumvent this limitation. We propose that the stronger His-Fe(III) coordination enabled by such a linkage serves to markedly enhance the effective ligand field strength of His-18. Our studies with 1 and 2 suggest that a double covalent linkage in cyt c may also enable energetically more favorable trans ligation of Met-80 than would be possible if only a single linkage were present. This would serve to further increase the stability of the protein fold and perhaps to increase the effective ligand field strength of Met-80 as well.     

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

The molecular structures of ferri- and ferrocytochrome c₅₅₁ from Pseudomonas aeruginosa have been refined at a resolution of 1.6 Å, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F_obs, and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state.Pseudomonas cytochrome c₅₅₁ differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c. As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c₅₅₁ than in cytochrome c.     

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Studies on the reaction of imidazole with cytochrome c3 from Desulfovibrio vulgaris

1. Cytochrome c₃, a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c₃ complex is detectable at 77 but not at 298 K.2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c₃ complex reflects a unique event for cytochrome c₃.3. Formation of the imidazole · cytochrome c₃ complex is strongly dependent on imidazole concentration (apparent K_d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c₃ spectral complex implicates ionization of an imidazole function (pK = 8.5).4. EPR studies at 8.5 K of ferricytochrome c₃ before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals.5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c₃ · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c₃.     

The reactions of octaethylporphyrin and its iron (II) and iron (III) complexes with free radicals

The reactions of dilute solutions of octaethylporphyrin and its iron (II) and iron (III) complexes with methyl, 2-cyanopropyl, t-butoxy, and benzoyloxy radicals are described. The results are summarized: (i) The reactivity of the porphyrin and its high-spin iron (II) and iron (III) complexes toward alkyl and t-butoxy radicals stands in the order: Fe^II > Fe^III ? free porphyrin. For benzoyloxy radicals the order is Fe^II > Porp > Fe^III. (ii) The exclusive path of reaction of high-spin iron (II) porphyrin with radicals is the rapid reduction of the radical and generation of an iron (III) porphyrin. The dominant path of reaction of high-spin iron (III) porphyrin with alkyl and (presumably) t-butoxy radicals is a rapid axial inner sphere reduction of the porphyrin. An axial ligand of iron is transferred to the radical. (iv) The reaction of benzoyloxy radicals with high or low-spin iron (III) porphyrins occurs primarily at the meso position. With the low-spin dipyridyl complex in pyridine the attendant reduction to iron (II) can be observed spectrally. Methyl radicals also reduce this complex by adding to the meso position. (v) The reaction of a radical with either an iron (II) or an iron (III) porphyrin results in the generation of the other valence state of iron and consequently oxidation and reduction products emanating from both iron species are obtained. (vi) No evidence for an iron (IV) is intermediate is apparent. (vii) Iron (II) porphyrins in solvents that impart either spin state are easily oxidized by diacyl peroxides. The occurrence of both axial and peripheral redox reactions with the iron complexes supports an underlying premise of a recent theory of hemeprotein reactivity. The relevance of the work to bioelectron transfer and heme catabolism is noted.     

Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis

The first investigation of the dynamics of a redox transition of an electron-transfer enzyme by time-resolved resonance Raman spectroscopy in combination with pulse-radiolytical reduction is described by an application to cytochrome c. A long-lived transient state is observed upon reduction of the alkaline form of cytochrome c as a distinct frequency shift of one resonance Raman band. From the frequency in the stable oxidized state, 1567 cm^?1, this particular resonance Raman band shifts within less than 1 μs to 1533 cm^?1 in the transient reduced state, which has a lifetime longer than 20 ms but shorter than a few seconds. Finally, in the stable reduced state, this band is located at 1547 cm^?1. According to a previous normal coordinate analysis, this resonance Raman band can be assigned predominantly to a stretching mode of the outermost C-C bonds in the four pyrrole rings of porphyrin. This vibrational mode is influenced by the protein most directly through the covalent thioether linkages of two cysteines to porphyrin. We interpret the long lifetime of the transient state as due to the slow return of Met-80 as sixth ligand to the heme iron upon reduction of the alkaline form of cytochrome c.     

Photodissociable endogenous ligand in alkaline-reduced cytochrome c peroxidase implicates distal protein tension

Laser excitation of alkaline- (pH 8.5) reduced cytochrome c peroxidase (CCP) produces resonance Raman (RR) bands arising from both low- and high-spin heme species (nu 3 = 1493/1471 cm-1) even though in the absence of laser excitation the absorption spectrum is characteristic of a purely low-spin species. The high-spin fraction is higher in a stationary than in a rotating sample, indicating that the high-spin contribution arises from photolysis induced by the Raman laser. This conclusion was confirmed by monitoring the absorption spectrum during laser irradiation. Photolability of the low-spin form is somewhat less than that of the CO adduct. The endogenous photolabile ligand is proposed to be the distal histidine residue, His-52. Recent picosecond absorption measurements (Jongeward et al., 1988) show that imidazole ligands in heme proteins do photodissociate but recombine in picoseconds, leading to net photostability on longer time scales. It is proposed that a fraction of the His-52 residues recombine much more slowly in CCP because of protein strain in the ligated form. This strain can also explain the anomalously rapid rate of CO binding to alkaline CCP.     

Kinetic studies on the effect of the heme iron(III) on the protein folding of ferricytochrome c.

The three-dimensional conformation of ferricytochrome c results from specific folding of the polypeptide chain around the covalently bound heme so that His-18 and Met-80 are axially coordinated to the Fe(III). The Fe(III)-free, porphyrin protein has an intrinsic viscosity, sedimentation coefficient, and circular dichroism indicative of a compact, globular protein conformation comparable to the holoprotein. Both the porphyrin protein and ferricytochrome c are reversibly denatured by guanidinium chloride. Refolding of the porphyrin protein occurs in essentially a single, exceptionally rapid kinetic phase (tau = 14 ms, 0.75 M guanidinium chloride, pH 6.5, 25 degrees C); whereas refolding of ferricytochrome c occurs in two slower kinetic phases (TAU 1 = 0.10 S, TAU 2 = 20 S) UNDER COMPARABLE CONDITIONS. The presence of Fe(III) in the metalloporphyrin of ferricytochrome c thus has a major effect on the protein folding kinetics. The slow kinetic phase is evidently due to this effect of Fe(III) and not to the slow cis-trans isomerism of the peptide bond of proline residues as has been suggested.     

Mechanisms of Mitochondrial Holocytochrome c Synthase and the Key Roles Played by Cysteines and Histidine of the Heme Attachment Site,Cys-XX-Cys-His

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c₁. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.     

Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration

The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the F^III and F^II CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pK _a values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of F^II, with F^III being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, F^II CT-cytochrome c being the most deranged.     

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236?±?3?mV in the absence of PEG, which was negatively shifted to +200?±?4?mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0?Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.     

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.     

YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50?kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be ?170?mV, +133?mV and +210?mV, respectively, at pH?7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH?7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M?=?0.6?±?0.2 and 1.8?±?0.5?mM H₂O₂, respectively), with similar turnover numbers (k_cat?=?19?±?2 and 13?±?2?s^?1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV)?=?O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E _m7 = –254 mV) and one low-spin, high-potential (E _m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c ₅₅₅ as the electron donor, the K _M and V _max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c ₅₅₅ oxidized min^–1 (mole cytochrome c peroxidase)^–1, respectively. Received: 6 January 1997 / Accepted: 27 May 1997     

Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode