Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer.

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymatic trimethylation of lysine-72 in cytochrome c

The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowering the pI values of the protein than the chemical modification. Furthermore, the lowering of the pI value of cytochrome c by enzymatic methylation is highly dependent on the urea concentration. The presence of urea reduces the effect of methylation on the protein molecule and the difference in pI values virtually disappears with the increasing concentration of urea (6 M), which essentially disrupts the protein tertiary structure.     

Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.     

Protein engineering of cytochrome c by semisynthesis: substitutions at glutamic acid 66

We have used protein semisynthesis to prepare four analogues of horse cytochrome c, in which the glutamic acid residue at position 66 has been removed and replaced by norvaline, glutamine, lysine and, as a methodological control, glutamic acid. This residue is quite strongly conserved in mitochondrial cytochrome c, and forms part of a cluster of acidic residues that occurs in all cytochromes c but whose function is obscure. Comparative studies of the physical and biochemical properties of the analogues have now disclosed two specific roles for Glu66 in the protein. It contributes significantly to the stabilization of the active conformation of the protein, probably by salt bridge formation, and it appears to participate in the redox-state-dependent ATP-binding site of cytochrome c. Our results also support two general views of the role of surface charged residues in cytochrome c, namely that their disposition influences both redox potential, through the electrostatic field felt at the redox centre, and the kinetics of electron transfer, through the dipole moment they generate.     

Nitrite reductase activity of cytochrome c

Small increases in physiological nitrite concentrations have now been shown to mediate a number of biological responses, including hypoxic vasodilation, cytoprotection after ischemia/reperfusion, and regulation of gene and protein expression. Thus, while nitrite was until recently believed to be biologically inert, it is now recognized as a potentially important hypoxic signaling molecule and therapeutic agent. Nitrite mediates signaling through its reduction to nitric oxide, via reactions with several heme-containing proteins. In this report, we show for the first time that the mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. Further, we demonstrate that in the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration. These data suggest an additional role for cytochrome c as a nitrite reductase that may play an important role in regulating mitochondrial function and contributing to hypoxic, redox, and apoptotic signaling within the cell.     

Tissue-specific differences between heart and liver cytochrome c oxidase

Bovine liver cytochrome c oxidase has been isolated and the subunit structure of this preparation compared with that of the bovine heart enzyme. Of the 10 nuclear-coded subunits, 3 were different in the 2 tissue forms, having different migrations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, different antigenicities to antibodies made against the heart subunits, and different N-terminal amino acid sequences. Subunit ASA of heart begins with the N-terminal sequence of SSG in liver and is different in 17 of the first 33 residues including a deletion of 2 residues in the liver isoform of this subunit. Subunit CVII of liver differs from its heart counterpart in 6 of the first 37 residues while subunit CIX from liver differs from the heart isoform in 15 of the first 25 residues. No differences between tissue types were observed in partial sequencing of the remaining nuclear-coded subunits. Recently, the major portion of the sequence of subunit CIX from rat liver has been obtained by cloning and sequencing of the cDNA for this polypeptide [Suske, G., Mengel, T., Cordingley, M., & Kadenbach, B. (1987) Eur. J. Biochem. 168, 233-237]. There is a greater sequence homology of the rat and bovine liver forms of CIX than there is between the bovine heart and liver isoforms.     

Ascorbate peroxidase activity of cytochrome c

Abstract

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.     

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.     

Protein matrix and dielectric effect in cytochrome c

The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, Delta E(m)(0)' can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. The evaluation of an apparent dielectric constant (U(exp)/U(theo)) yields variable, and sometimes even negative, values if U(exp) = Delta G(0)redox. However, some consistent result are observed if U(exp) = Delta H(0)redox, with a measured epsilon(Delta Delta)(H)(redox) = 19 +/- 6. The variability is thus attributed to an entropic factor (epsilon(Delta Delta)(S)(redox)) that is investigated using a series of substitutions of Asn(52) and/or Tyr(67). In double mutants Y67F/N52I Y67F/N52V, where most of the hydrogen bond network in the heme crevice is eliminated, Delta S(redox) compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.     

Monooxygenase activity of cytochrome c peroxidase.

Recombinant cytochrome c peroxidase (CcP) and a W51A mutant of CcP, in contrast to other classical peroxidases, react with phenylhydrazine to give sigma-bonded phenyl-iron complexes. The conclusion that the heme iron is accessible to substrates is supported by the observation that CcP and W51A CcP oxidize thioanisole to the racemic sulfoxide with quantitative incorporation of oxygen from H2O2. Definitive evidence for an open active site is provided by stereoselective epoxidation by both enzymes of styrene, cis-beta-methylstyrene, and trans-beta-methylstyrene. trans-beta-methylstyrene yields exclusively the trans-epoxide, but styrene yields the epoxide and phenylacetaldehyde, and cis-beta-methylstyrene yields both the cis- and trans-epoxides and 1-phenyl-2-propanone. The sulfoxide, stereoretentive epoxides, and 1-phenyl-2-propanone are formed by ferryl oxygen transfer mechanisms because their oxygen atom derives from H2O2. In contrast, the oxygen in the trans-epoxide from the cis-olefin derives primarily from molecular oxygen and is probably introduced by a protein cooxidation mechanism. cis-[1,2-2H]-1-Phenyl-1-propene is oxidized to [1,1-2H]-1-phenyl-2-propanone without a detectable isotope effect on the epoxide:ketone product ratio. The phenyl-iron complex is not formed and substrate oxidation is not observed when the prosthetic group is replaced by delta-meso-ethylheme. CcP thus has a sufficiently open active site to form a phenyl-iron complex, to oxidize thioanisole to the sulfoxide, and to epoxidize styrene and beta-methylstyrene. The results indicate that a ferryl (Fe(IV) = O)/protein radical pair can be coupled to achieve two-electron oxidations. The unique ability of CcP to catalyze monooxygenation reactions does not conflict with its peroxidase function because cytochrome c is oxidized at a distinct surface site (DePillis, G. D., Sishta, B. P., Mauk, A. G., and Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19334-19341).     

The influence of amino acid substitutions on the conformational energy of cytochrome c.

Conformational energies have been evaluated for each of the staggered side-chain conformations associated with the 261 amino acid substitutions known to occur among 60 eucaryotic species. At least 86% of these substitutions can be sterically accommodated (one at a time) within the structure of horse-heart cytochrome c resulting from conformational energy refinement. Simultaneous incorporation of all pertinent amino acid substitutions found in eight representative species into the refined horse-heart structure is also shown to be sterically possible, with few exceptions. In two cases (Pekin duck cytochrome with 10 substitutions and Samia cynthia cytochrome with 24 substitutions), all substitutions could be readily incorporated, and the total energies associated with their computed structures differed by less than 10 kcal/mol from that of horse-heart cytochrome c. In the cytochromes from rattlesnake (22 substitutions), tuna (18 substitutions), and Neurospora crassa (36 substitutions), tyrosine could not be substituted for phenylalanine at position 46, within the constraints of the calculations. However, when all of the remaining substitutions were incorporated into these three cytochromes, their computed conformational energies differed by less than 30 kcal/mol from that of horse-heart cytochrome c. Between two and four amino acid substitutions cause high energies in the cytochromes from human, baker's yeast, and cotton seed, but all of the remaining substitutions are consistent with a low energy conformation. These results suggest that the structures of homologous proteins may be even more similar than has previously been recognized. Substitutions of all possible amino acid types at the invariant positions (where all eucaryotic cytochromes c bear the same amino acid) have revealed some cases where different amino acids can be accommodated, thus demonstrating that the biological constraints on amino acid substitutions are often different from the purely steric constraints investigated in this work.     

Antihypoxic activity of cytochrome c heme peptides

A comparative study of antihypoxic activity of five and two cytochrome c derivatives was performed during their single prophylactic administration on the model of acute hypobaric hypoxia (AHBH) and during rehabilitation period after AHBH, respectively. Antihypoxic efficiency of cytochrome c derivatives was shown to be dependent on doses, time of drug administration, and type of experimental animal resistance. The heme-nonapeptide of cytochrome c proved to be of maximum efficiency during prophylactic administration and rehabilitation period after AHBH.     

Reduction and activity of cytochrome c in the cytochrome c-cytochrome aa3 complex.

An interaction between rat liver glucocorticoid--receptor complex and immobilized ATP was identified. Rat liver cytosol preparations were incubated with [3H]triamcinolone acetonide for 4 h at 4 degrees C and partially purified by precipitation with (NH4)2SO4 before use. The resulting glucocorticoid--receptor complex could be selectively adsorbed on to columns of ATP--Sepharose. The freshly prepared cytosol [3H]triamcinolone acetonide--receptor complex had very little affinity for binding to the ATP--Sepharose column, but acquired this ability on temperature- or salt-activation. The presence of 10 mM-sodium molybdate during this salt- or temperature-dependent activation blocked the binding of the receptor complex to ATP--Sepharose. The interaction is reversible, since it can be disrupted by high-salt conditions. A competitive binding assay, using free nucleotides in samples to be chromatographed, revealed a preferential interaction between ATP and the glucocorticoid--receptor complex. Buffer containing ATP was also used to elute the glucocorticoid--receptor complex from ATP--Sepharose columns successfully. When ATP was added to the preparations containing [3H]triamcinolone acetonide--receptor complexes, the steroid specificity or sedimentation properties of the complex remained unaltered. Our results demonstrate an interaction between rat liver glucocorticoid--receptor complex and immobilized ATP and suggest a role of this nucleotide in receptor function.