Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.     

Conservation of the free energy change of the alkaline isomerization in mitochondrial and bacterial cytochromes c

The thermodynamic parameters of the alkaline transition for oxidized native yeast iso-1 cytochrome c and Rhodopseudomonas palustris cytochrome c(2) (cytc(2)) have been determined through direct electrochemistry experiments carried out at variable pH and temperature and compared to those for horse and beef heart cytochromes c. We have found that both transition enthalpy and entropy are remarkably species dependent, following the order R. palustris cytc(2) > beef (horse) heart cytc>yeast iso-1 cytc. Considering the high homology at the heme-protein interface in the native species, this variability is likely to be mainly determined by differences in the structural and solvation properties and the relative abundance of the various alkaline conformers. Notably, changes in transition enthalpy and entropy among these cytochromes c are compensative and result in small variations in the free energy change of the process (which amounts approximately to +50 kJ mol(-1)) and consequently in the apparent pK(a) value. This compensation indicates that solvent reorganization effects play an important role in the thermodynamics of the transition. This mechanism is functional to ensure a relatively high pK(a) value for the alkaline transition, which is needed to preserve His,Met ligation to the heme iron in cytochrome c at physiological pH and temperature, hence the E(o) value required for the biological function.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.     

Protective role of mitochondrial unsaturated lipids on the preservation of the apoptotic ability of cytochrome C exposed to singlet oxygen

Cytochrome c-mediated apoptosis in cells submitted to photodynamic therapy raises the question about the ability of photodynamically oxidized cytochrome c (cytc405) to trigger apoptosis as well as the effect of membranes on protein photo-oxidation. Cytochrome c was submitted to irradiation in the presence of MB+ in phosphate buffer and in the presence of four types of phosphatidylcholine/phosphatidylethanolamine/cardiolipin (PCPECL) liposomes (50/30/20%): totally saturated lipids (tsPCPECL), totally unsaturated lipids (tuPCPECL), partially unsaturated (80%) lipids, with unsaturation in the PC and PE content (puPCPECL80), and partially unsaturated (20%) lipids, with unsaturation in the CL content (puPCPECL20). Cytc405 was formed by irradiation in buffered water and in tsPCPECL and puPCPECL20 liposomes. In the presence of tuPCPECL and puPCPECL80, cytochrome c was protected from photodynamic damage (lipid-protected cytochrome c). In CL liposomes, 25% unsaturated lipids were enough to protect cytochrome c. The presence of unsaturated lipids, in amounts varying according to the liposome composition, are crucial to protect cytochrome c. Interesting findings corroborating the unsaturated lipids as cytochrome c protectors were obtained from the analysis of the lipid-oxidized derivatives of the samples. Native cytochrome c, lipid-protected cytochrome c, and cytc405 were microinjected in aortic smooth muscle cells. Apoptosis, characterized by nucleus blebbing and chromatin condensation, was detected in cells loaded with native and lipid protected cytochrome c but not in cells loaded with cytc405. These results suggest that photodynamic therapy-promoted apoptosis is feasible due to the protective effect of the mitochondrial lipids on the cytochrome c structure and function.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

Kinetic aspects of the interaction of horse heart cytochrome c with sodium dodecyl sulfate

The sodium dodecyl sulfate (SDS) concentration dependence of spectral changes in circular dichroism (CD) and in absorbance of cytochrome c were examined in the far-ultraviolet region, aromatic region, and the Soret band. The Soret peak obtained in 0.60 mM SDS was nine times greater than that of the native state. (The critical micelle concentration, CMC, of SDS was 2.2 mM in the phosphate buffer used.) The results indicated that the drastic change at the Soret band did not accompany the corresponding large-scale change in secondary structure of the protein. In the stopped-flow measurements, two and three processes were followed at 406 nm below and above the CMC, respectively. At 289 nm only one process was observed, and this corresponded to the second process at 406 nm. Therefore, the second process at 406 nm was considered to be a change in tertiary structure around the heme group. The first process and the third process seemed to reflect a change in the heme environment; the former appeared to be due to a solvent effect and the latter due to a binding effect of a large number of dodecyl sulfate ions.     

Methionine sulfoxide cytochrome c.

Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs during the photooxidation reaction. The absorbance spectrum of methionine sulfoxide ferricytochrome c at neutrality is similar to that of the unmodified protein except for an increase in the extinction coefficient of the Soret absorbance band and for the complete loss of the ligand sensitive 695 nm absorbance band in the spectrum of the derivative. The protein remains in the low spin configuration which implies the retention of two strong field ligands. Spin state sensitive spectral titrations and model studies of heme peptides indicate that the sixth ligand is definitely not provided by a lysine residue but may be methionine-80 sulfoxide coordinated via its sulfur atom. Circular dichroism spectra indicate that the heme crevice of methionine sulfoxide ferri- and ferrocytochrome c is weakened relative to native cytochrome c. The redox potential of methionine sulfoxide cytochrome c is 184 mV which is markedly diminished from the 260 mV redox potential of native cytochrome c. The modified protein is equivalent to native cytochrome c as a substrate for cytochrome oxidase and is not autoxidizable at neutral pH but is virtually inactive with succinate-cytochrome c reductase. These results indicate that the major role of the methionine-80 in cytochrome c is to preserve a closed hydrophobic heme crevice which is essential for the maintainance of the necessary redox potential.     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

Folding character of cytochrome c studied by o-nitrobenzyl modification of methionine 65 and subsequent ultraviolet light irradiation

The protein folding character of cyt c was studied with the use of a photocleavable o-nitrobenzyl derivative of Met65 (NBz-Met65). For the NBz-Met65 cyt c, the Soret absorption band slightly blue shifted compared with the unlabeled cyt c, the 695 nm absorption band related to the Met80 sulfur ligation to the heme iron disappeared, and its resonance Raman spectrum was characteristic of a six-coordinate low-spin species, all characters demonstrating coordination of a non-native ligand, probably a histidine, instead of Met80 to the heme iron. The far-UV circular dichroism (CD) spectrum of cyt c was altered, and the transition midpoint concentration value of guanidine hydrochloride (GdnHCl) for unfolding the protein decreased by 0.9 M by the modification, which showed perturbation of the structure and decrease in protein stability, respectively. With irradiation of 308 nm laser pulses on the NBz-Met65 cyt c, the Soret absorption band slightly red shifted, the 695 nm absorption band appeared, and the CD spectrum shifted toward that of the native protein, which demonstrated recovery of the methionine heme coordination and the native protein structure, due to reconversion of NBz-Met65 to unlabeled methionine. A fast phase was detected as a change in Soret absorbance with a rate constant of 21 000 +/- 4000 s(-)(1) during refolding of cyt c initiated by irradiation of a 308 nm pulse on the NBz-Met65 cyt c in the presence of 2 M GdnHCl. The observed rate constant corresponded well with that reported by the tryptophan fluorescence study [Shastry, M. C. R. S., and Roder, H. (1998) Nat. Struct. Biol. 5, 385-392]. The intermediate decayed with a rate constant of 90 +/- 15, followed by another phase with a rate constant of 13 +/- 3 s(-)(1), and was not seen in the absence of GdnHCl.     

Mechanism of activation of cytochrome c peroxidase activity by cardiolipin

In this work, the actions of bovine heart cardiolipin, synthetic tetraoleyl cardiolipin, and a nonspecific anionic detergent sodium dodecyl sulfate (SDS) on cytochrome c (Cyt c) peroxidase activity recorded by chemiluminescence in the presence of luminol and on the Fe...S(Met80) bond whose presence was estimated by a weak absorption band amplitude with peak at 695-700 nm (A(695)) were compared. A strict concurrency between Fe...S(Met80) breaking (A(695)) and cytochrome peroxidase activity enhancement was shown to exist at cardiolipin/Cyt c and SDS/Cyt c molar ratios of 0 : 1 to 50 : 1 (by chemiluminescence). Nevertheless, when A(695) completely disappeared, Cyt c peroxidase activity under the action of cardiolipin was 20 times more than that under the action of SDS, and at low ligand/protein molar ratios (=4), SDS failed to activate peroxidase activity while cardiolipin enhanced Cyt c peroxidase activity 16-20-fold. A(695) did not change on Cyt c binding with liposomes consisting of tetraoleyl cardiolipin and phosphatidylcholine (1 : 10 : 10), while peroxidase activity was enhanced by a factor of 8. Breaking of 70% of the Fe...S(Met80) bonds resulted in only threefold enhancement of peroxidase activity. Cardiolipin-activated Cyt c peroxidase activity was reduced by high ionic strength solution (1 M KCl). The aggregated data suggest that cardiolipin activating action is caused, first, by a nonspecific effect of Fe...S(Met80) breaking as the result of conformational changes in the protein globule caused by the protein surface electrostatic recharging by an anionic amphiphilic molecule, and second, by a specific acceleration of the peroxidation reaction which is most likely due to enhanced heme accessibility for H(2)O(2) as a result of the hydrophobic interaction between cardiolipin and cytochrome.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation

To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Molecular architecture of cytochrome oxidase and its transition on treatment with alkali or sodium dodecyl sulfate.

In dimeric cytochrome oxidase [EC 1.9.3.1], one of the two heme a molecules of one monomeric unit has been proposed to be converted by the other unit, thus becoming latent in terms of catalytic functions (1). As the dimer was split into two monomers by treatment with alkali or sodium dodecyl sulfate (SDS), it was shown that the intensity of circular dichroism (CD) in the Soret region due to heme a decreased, probably reflecting release of the strain on the latent heme. On the other hand, the profile of magnetic circular dichroism (MCD) was nearly unchanged during this conversion, except for a weakening of the signal due to deprotonation of the heme during the alkali treatment. When the monomer was further dissociated into constituent subunits in strong alkali or at high concentrations of SDS, the CD spectrum disappeared almost completely, indicating loss of the asymmetric interactions of the chromophoric heme a with its immediate environments, consisting of the subunit assembly. The MCD pattern also suffered a small change as the dissociation proceeded, and a specific pattern appeared as the Schiff base was finally formed. The Schiff base formation of cytochrome oxidase in strong alkali proceeded in two steps whether the heme iron was in the oxidized or reduced state. As a consequence of the initial rapid reaction, the enzyme was suggested to have been disintegrated into constituent subunits with heme a being attached nonspecifically to either one, and structural characteristics dependent on the redox state were completely lost. The Arrehenius plot for this rapid change showed a break, indicating a transition in the structure of the cytochrome oxidase assembly, although no such phenomenon was observed during the slow reaction. Activation parameters in the rapid and slow reactions for the oxidized and reduced oxidase are given. Based on these findings, as well as other considerations, a molecular architecture of this enzyme is proposed; the role of heme a in anchoring four 14,000-dalton polypeptides into the minimal functional unit catalyzing the aerobic oxidation of ferrocytochrome c is emphasized.     

Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin

Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cooperative binding of hexadecyltrimethylammonium chloride and sodium dodecyl sulfate to cytochrome c and the resultant change in protein conformation

The binding of hexadecyltrimethylammonium chloride (HTAC) and sodium dodecyl sulfate (SDS) to cytochrome c was determined by potentiometric titration and the corresponding changes in protein conformation by circular dichroism (CD). The binding isotherms were biphasic; about 20 surfactant cations or anions were bound to cytochrome c in the first phase. Another 30 or so HTA+ ions were bound in the second phase, which was below the critical micelle concentration of the surfactant, but the binding of dodecyl sulfate ions in the second phase increased sharply near the critical micelle concentration. The binding of both surfactants was highly cooperative and was endothermic; the data in the first phase fitted the Hill plot. The corresponding change in the secondary structure of cytochrome c was small; the CD spectra in the ultraviolet region showed a moderate increase in the helicity in HTAC solution and some changes in conformation in SDS solution. However, the CD spectra for the Soret band indicated a marked change in the local conformation around the heme.     

Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.     

Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.     

Resonance Raman spectroscopy of cytochrome oxidase using Soret excitation: selective enhancement, indicator bands, and structural significance for cytochromes a and a3

Resonance Raman studies of oxidized and reduced cytochrome oxidase and liganded derivatives of the oxidized enzyme have been performed by using direct-Soret excitation at 413.1 and 406.7 nm, as well as near-Soret excitation (457.9 nm) and alpha-band excitation (604.6 nm). The Soret results clearly show selective enhancement of Raman modes of the hemes of cytochromes a and a3, depending upon the excitation wavelength chosen. For the preparations employed in this study, photoreduction of cytochrome oxidase in the laser beam was not a significant problem. Resonance Raman frequencies sensitive to oxidation state and spin state or core expansion of the a and a3 hemes are identified and correlated with those previously identified for other heme proteins. An unusual low-frequency (less than 500 cm(-1)) spectrum is observed for oxidized high-spin cytochrome a3, which may be due to axial nonheme structures in this cytochrome.     

Conformational changes in cytochrome c and cytochrome oxidase upon complex formation: a resonance Raman study

The fully oxidized complex of cytochrome c and cytochrome oxidase formed at low ionic strength was studied by resonance Raman spectroscopy. The spectra of the complex and of the individual components were compared over a wide frequency range using Soret band excitation. In both partners of the complex, structural changes occur in the heme groups and in their immediate protein environment. The spectra of the complex in the 1600-1700 cm-1 frequency range were dominated by bands from the cytochrome oxidase component, whereas those in the 300-500 cm-1 range were dominated by bands from the cytochrome c component, hence allowing separation of the contributions from the two individual species. For cytochrome c, spectral changes were observed which correspond to the induction of the conformational state I and the six-coordinated low-spin configuration of state II on binding to cytochrome oxidase. While in state I the structure of cytochrome c is essentially the same as in solution, state II is characterized by a structural rearrangement of the heme pocket, leading to a weakening of the axial iron-methionine bond and an opening of the heme crevice which is situated in the center of the binding domain for cytochrome oxidase. The relative contributions of the two cytochrome c states were estimated to be approximately in the ratio 1:1 in the complex.(ABSTRACT TRUNCATED AT 250 WORDS)