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Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

A cytochrome-c (cyt c) oxidase supercomplex consisting of 7-8 subunits and possessing a mass of 358-425 kDa was purified from Bradyrhizobium japonicum bacteroid membranes. At least two subunits possess c-type heme as a prosthetic group. One of the c-heme-containing components was detected in bacteroid membranes, but not in free-living cells. The complex also contains b-heme, and both b-type and c-type heme proteins were spectrophotometrically shown to form complexes with carbon monoxide. A CO difference spectrum showed an absorption minimum (trough) at 551.7 nm, possibly corresponding to a previously described cyt c-552 in bacteroid membranes. 1 mM quinacrine (Atebrin) had no effect on O2 uptake by the cytochrome-c oxidase complex, but 10 mM inhibited O2 uptake by 90%. Cytochromes b and c1 of the cytochrome bc1 respiratory complex were identified as two of the components of the bacteroid complex based upon immunoreaction with antibodies against these two proteins from B. japonicum. The oxidase complex oxidized exogenously added horse heart ferrocytochrome c concomitant with the uptake of oxygen. It could also oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine in the absence of added cytochrome c. Oxygen uptake activity was completely inhibited by 10 microM NaCN and 38% by 0.1 microM NaCN. The oxidase complex was not able to oxidize a ubiquinol homolog possessing a single isoprenoid unit side chain. Solubilization of bacteroid membranes in the presence of 1.0 mM EDTA resulted in complete loss of cytochrome-c oxidase activity. Leghemoglobin deoxygenation data indicated that the oxidase complex can efficiently function at free oxygen concentrations well below 1.0 microM, even though attempts to determine the oxidase's specific affinity oxygen were unsuccessful due to the formation of oxidized leghemoglobin derivatives.

Proteinase K was used to degrade membrane proteins exposed at the outer (cytoplasmic) and inner (periplasmic) surface of sealed, uniformly oriented chromatophore vesicles of Rhodobacter sphaeroides. Exclusive and controlled digestion of the chromatophore interior was achieved after Ca(2+)-induced fusion with large unilamellar phosphatidylglycerol liposomes containing microencapsulated enzyme. Reaction center subunit H, which served as a marker for the outer surface, was degraded to a slightly smaller product in chromatophores. This protein remained intact after liposome-chromatophore fusion, suggesting that the intermixing of lipid bilayers proceeded without significant leakage of the aqueous vesicle contents. In contrast, while cytochrome c1 was not affected in chromatophores, 70-75% was degraded within 60 min after liposome-chromatophore fusion. These results support an arrangement in which the bulk of this protein, including the mesoheme component and active site residues, faces the periplasmic side of the membrane. Although current functional models for the cytochrome bc1 complex predict that the Rieske iron-sulfur center interacts with cytochrome c1 in the periplasm, the iron-sulfur protein resisted proteolytic attack in the liposome-chromatophore fusion products under conditions that caused extensive degradation of cytochrome c1. Two cleavage products of the iron-sulfur protein were observed after the digestion of chromatophores, suggesting both a heterogeneity in the population of this protein and the exposure of at least part of its molecular mass to the cytoplasm.

The effects of iron deficiency on the aerobic pathway of energy metabolism were studied using mitochondria isolated from epithelial cells from the hamster cheek pouch. A statistically significant reduction in the concentrations of cytochromes aa3, b and c (P less than 0.05), a reduction (P = 0.064) in cytochrome cl and altered cytochrome ratios were found in the mitochondria of iron deficient compared to normal animals. State 4 respiration was demonstrated in the mitochondria of both normal and iron deficient animals but state 3 respiration could not be demonstrated; this suggests uncoupling of oxidative phosphorylation which may be an artefact associated with the separation of epithelium from its connective tissue. Nevertheless we conclude that the reduction in cytochrome concentration is a real effect of iron deficiency which may explain, at least in part, the reduction of both energy production and cell proliferation seen in oral epithelia under these conditions.

Phospholipids in isolated Complex III of the mitochondrial electron-transfer chain were depleted by hydrophobic chromatography. The complex was further purified by affinity chromatography. The polypeptide composition of the complex was examined using SDS-polyacrylamide gel electrophoresis. Ten polypeptides were demonstrated in the gel pattern of the complex containing more than 10% (w/w) phospholipids; and 9 polypeptides, in the pattern of the complex containing 5% phospholipids. Although the enzymic activity of the complex composed of the 9 polypeptides was about a half of that of the original enzyme, it was fully restored when soybean phospholipid mixture was added. Further depletion of phospholipids to 0.6% makes the iron-sulfur protein dissociable from the complex, resulting in a loss of the enzymic activity (Shimomura, Y. and Ozawa, T. (1982) Biochem. Int. 5, 1-6). These results suggest that Complex III consists of 9 polypeptides, and the smallest polypeptide is a contaminant embedded in phospholipids with respect to the electron-transfer capability of the complex.

A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible.

A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.

A method is described for characterizing, c-type cytochromes in bacterial membrane preparations according to molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Applied to the photosynthetic bacterium Rhodopseudomonas sphaeroides this technique is used, together with spectroscopic measurements, to demonstrate that a membrane-bound cytochrome c of mol.wt. 30000 is active in photosynthetic electron transport in addition to the well-known soluble cytochrome, cytochrome c2. The membrane cytochrome has a midpoint potential (E'0) at pH 7 of +290 mV, as compared with +360 mV for purified cytochrome c2. Its alpha-band has a peak near 552 nm, as compared with 550 nm for cytochrome c2. Evidence is presented that chromatophores contain roughly equal amounts of the two cytochromes.

The preparation, purification, and characterization of four new derivatives of cytochrome c trifluoroacetylated at lysines 72, 79, 87, and 88 are reported. The redox reaction rates of these derivatives with cytochrome b5, cytochrome c1 and cytochrome oxidase indicated that the interaction domain on cytochrome c for all three proteins involves the lysines immediately surrounding the heme crevice. Modification of lysines 72, 79, 87 had a large effect on the rate of all three reactions, while modification of lysine 88 had a very small effect. Even though lysines 87 and 88 are adjacent to one another, lysine 87 is at the top left of the heme crevice oriented towards the front of cytochrome c, while lysine 88 is oriented more towards the back. Since the interaction sites for cytochrome c1 and cytochrome oxidase are essentially identical, cytochrome c probably undergoes some type of rotational diffusion during electron transport.

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.

The primary structure of the cytochrome c1 subunit of ubiquinol-cytochrome-c reductase from mitochondria of Neurospora crassa was determined by sequencing the cDNA of a bank cloned in Escherichia coli. From the coding region the sequence of 332 amino acids, corresponding to the molecular mass of 36,496 Da, was derived for the precursor protein. The mature protein, the N terminus of which was previously sequenced [Tsugita et al. (1979) in Cytochrome oxidase (King, T. E. et al., eds) pp. 67-77, Elsevier, New York], consists of 262 amino acids and has the molecular mass of 29,908 Da including the heme. The sequence contains an N-terminal hydrophilic part of 211 residues, which carries the heme, a hydrophobic stretch of 15 residues, which is assumed to anchor the protein to the membrane, and a C-terminal hydrophilic part of 36 residues. The N-terminal presequence of 70 amino acids contains 9 positive charges but only 1 negative charge and is characterized by a stretch of 20 uncharged residues.

The autoxidizability of beef heart cytochrome c1 was investigated in terms of the integrity of the binding of the hinge protein to the heme subunit. Cytochrome c1 was isolated as a subcomplex consisting of the heme subunit and the hinge protein. Treatment of the cytochrome c1 subcomplex with p-chloromercuribenzoate (pCMB) under mild conditions lessened the binding strength between the two subunits. They were dissociated on polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions, but were not separated by gel filtration chromatography. The pCMB-treated subcomplex had a slight autoxidizability. This was repressed to the level of the native subcomplex, when the mercurial compound bound to the subcomplex was removed by the addition of 2-mercaptoethanol. Concomitantly, the less stable binding between the subunits was apparently reversed to the native state. After pCMB treatment of the subcomplex, the heme subunit recovered from PAGE showed marked autoxidizability, even if it was treated with 2-mercaptoethanol. Addition of cholate repressed the autoxidizability of the heme subunit after the removal of the mercurial compound. These results confirmed that the stable binding of the hinge protein to the heme subunit was essential for the nonautoxidizability of cytochrome c1 subcomplex. In addition, it was suggested that cysteinyl residues in the subcomplex must be involved to a great extent in the stable binding between the two subunits.

The complete primary structure of bovine heart cytochrome c1 was established by analyses of peptide fragments prepared by digestion using trypsin, staphylococcal protease, and chymotrypsin and by cyanogen bromide cleavage of cytochrome c1 and its derivatives. The total number of amino acid residues is 241, giving a molecular weight of 27,924 including the heme group. The NH2- and COOH-terminal residues are serine and lysine, respectively. One characteristic of the protein is that cytochrome c1 contains 43.6% hydrophobic residues and the polarity is estimated to be 41.1%. No clear homology was found between cytochrome c1 and other membranous proteins such as cytochrome b5 or the subunits of cytochrome oxidase for which sequences have been reported. Cytochrome c1 is predicted to have a high content of alpha-helix (46%). Partial sequence studies were also carried out on cytochrome c1 preparations obtained by different procedures and showed that there is no difference among the sequences of various preparations of cytochrome c1. The presence of a hydrophobic cluster near the COOH-terminal region indicates that the COOH-terminal region of cytochrome C1 associates with, or is buried in, the phospholipid bilayer of the mitochondrial membrane.

In several brain parts of rats with experimental neurosis local circulation rate (LCR) was measured by hydrogen clearence method, and cytochromes content was determined by differential spectrophotometry. In early period of neurotization (up to 15 days) reciprocal LCR changes were observed in the cortex and subcortical structures, and after 18 days were reduced in all the brain parts studied. The maximum reduction was observed after three weeks of neurotization. Neurotization during one week elicited significant decrease of cytochrome a level in the cortex. Its level in the cortex after 3 weeks of neurotization and in the hypothalamus and hippocampus after one and three weeks of neurotization did not differ from its normal content. Neurotization did not influence cytochromes c + c1 levels in the structures examined.

The content of mitochondrial protein and cytochromes aa3, b, c1 and c in rat liver and heart was determined in four groups of rats: controls, CAP-treated rats, exercised rats and CAP-treated exercised rats. Single swimming does not change the amount of mitochondrial protein in liver and myocardium as well as the content of cytochromes aa3, b, c1 and c in liver mitochondria. The content of cyt. c is lowered in heart mitochondria. CAP treatment of rats for three days decreases the amount of mitochondrial proteins and cyt. aa3, and b whereas no such changes are observed in heart mitochondria. The subsequent swimming on the background of the inhibitor does not affect additionally liver mitochondria, but lowers significantly the mitochondrial protein and the content of cyt. aa3, b, c1 and c in the myocardium. These data show that exhaustive muscular work alters the effect of CAP in heart mitochondria. Three hours of exhaustive swimming are not sufficient to change neither the amount of total mitochondrial protein, nor the cytochrome content. The lowered cyt. c content in heart mitochondria after swimming is explained by an intensified permeability of the outer membrane. Thus, the changes in the activity of some enzymes and the oxidative phosphorylation previously reported by us, are not due to alterations in the protein synthesis.

Room temperature near-infrared magnetic circular dichroism and low-temperature electron paramagnetic resonance measurements have been used to characterize the ligands of the heme iron in mitochondrial cytochromes c, c1, and b and in cytochrome f of the photosynthetic electron transport chain. The MCD data show that methionine is the sixth ligand of the heme of oxidized yeast cytochrome c1; the identify of this residue is inferred to be the single conserved methionine identified from a partial alignment of the available cytochrome c1 amino acid sequences. A different residue, which is most likely lysine, is the sixth heme ligand in oxidized spinach cytochrome f. The data for oxidized yeast cytochrome b are consistent with bis-histidine coordination of both hemes although the possibility that one of the hemes is ligated by histidine and lysine cannot be rigorously excluded. The neutral and alkaline forms of oxidized yeast cytochrome c have spectroscopic properties very similar to those of the horse heart proteins, and thus, by analogy, the sixth ligands are methionine and lysine, respectively.

A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.