Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.
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PMID:Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes. 165 82

We have characterized the activities of the cytochrome bc1 complex in mitochondrial membranes from a yeast strain in which we deleted the nuclear gene (QCR6, COR3) which codes for the highly acidic subunit 6 of the bc1 complex. The chromosomal copy of QCR6 was replaced with a plasmid derived copy of QCR6, in which the entire coding region of QCR6 was replaced with the yeast LEU2 gene. The resulting deletion strain, MES8, contained no detectable mRNA for QCR6, and the cytochrome bc1 complex purified from the deletion strain lacked subunit 6. The deletion strain respired and grew on nonfermentable carbon sources such as ethanol and glycerol. Ubiquinol-cytochrome c reductase activity of mitochondria from the deletion strain was decreased 50% under conditions where the activity is zero order with respect to cytochrome c, and there was a similar decrease in the first-order rate constant for cytochrome c reduction. The loss of bc1 complex activities, observed at physiological ionic strengths, was reversible. Both the zero order rate and the first-order rate constant for cytochrome c reduction could be recovered to those of the parental strain by measuring these activities in mitochondrial membranes under conditions of low ionic strength. The zero order rate and first-order rate constant for cytochrome c reduction in membranes from the parent, wild-type yeast showed essentially no change coincident with this change in ionic strength. The 50% drop in both turnover number and first-order rate constant of ubiquinol-cytochrome c reductase activity indicates that half of the cytochrome bc1 complexes are inactive in the deletion strain at physiological ionic strengths. Inhibition by myxothiazol of cytochrome c reductase activity of mitochondrial membranes from the deletion strain showed an ionic strength-dependent lag in the titration curve that extended to the point where half of the inhibitor sites are filled. This lag was not observed with membranes from the wild-type, parent strain. This response to the inhibitor is consistent with half of the cytochrome bc1 complexes being inactive in mitochondria from the deletion strain at physiological ionic strength, but with both active and inactive complexes still able to bind inhibitor. The reversible, half-of-the-sites reactivity indicates that the bc1 complex must be dimeric in situ, in agreement with previous findings that the complexes isolated from fungal (Leonard, K., Wingfield, P., Arad, T., and Weiss, H. (1981) J. Mol. Biol. 149, 259-274) and mammalian (Nalecz, M. J., Bolli, R., and Azzi, A. (1985) Arch. Biochem. Biophys. 236, 619-628) mitochondria are structural dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Subunit 6 regulates half-of-the-sites reactivity of the dimeric cytochrome bc1 complex in Saccharomyces cerevisiae. 217 Mar 63

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
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PMID:Identification of the binding site on cytochrome c1 for cytochrome c. 298 91

Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain.
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PMID:Functional domains of assimilatory NADH:nitrate reductase from Chlorella. 301 63

A method for the large-scale isolation of beef heart mitochondrial cytochrome c1 in high purity was developed. This method gave higher yield of "one-band" cytochrome c1 than previously reported [Kim, C. H., & King, T. E. (1981) Biochem. Biophys. Res. Commun. 102, 607-614]. In addition, the present method was effective in the preparation of "two-band" cytochrome c1 which was used to prepare the hinge protein according to the principle of sequential resolution [Kim, C. H., & King, T. E. (1983) J. Biol. Chem. 258, 13543-13551]. The isolation of one-band and two-band cytochrome c1 by this procedure could be completed within 3 or 4 days starting with succinate-cytochrome c reductase. One-band cytochrome c1 showed a molecular weight of 44,000 by sedimentation equilibrium and 29,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The disparities in these data from the actual value of 27,924 by amino acid sequence analysis, as previously reported [Wakabayashi, S., Matsubara, H., Kim, C. H., & King, T. E. (1982) J. Biol. Chem. 257, 9335-9344], are most probably due to the formation of detergent or detergent-phosphate complex. A comparison of some properties of one-band cytochrome c1 with those of two-band cytochrome c1 clearly showed significant differences between the two preparations. These results suggest the hypothesis that one of the possible roles of the hinge protein in the mitochondrial respiratory chain is to stabilize the conformation of cytochrome c1.
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PMID:Preparation and properties of cardiac cytochrome c1. 303 14

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
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PMID:Limited proteolysis of the nitrate reductase from spinach leaves. 319 46

We have determined the DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the ubiquinol-cytochrome c reductase. The reading frame found encodes a putative polypeptide of 17 394 daltons. This protein is highly unusual: 38% of its residues are acidic and 14% are basic amino acids. The most notable feature in the protein sequence is a stretch of 25 consecutive acidic amino acids. The polypeptide has homology with the 9-kd so-called 'hinge' protein of beef-heart complex III, which also has a cluster of acidic residues. Acidic amino acids are likely to be essential for the function of these proteins, since their degree of conservation is higher than that of other residues.
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PMID:The DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a protein with an extremely high content of acidic amino acids. 632 32

The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) is a nuclear-coded protein of the hydrophobic fragment of the enzyme. We have isolated and sequenced a full-length cDNA clone coding for this polypeptide. The deduced protein is 104 amino acid residues long with a molecular mass of 12305 Da. This particular subunit of complex I lacks a cleavable mitochondrial targeting sequence. In agreement with its localization within complex I, we have found that this subunit behaves like an intrinsic membrane protein. Nevertheless, the deduced protein is rather hydrophilic, exhibiting no hydrophobic domain long enough to traverse a membrane in an alpha-helical conformation. The 12.3 kDa subunit shows a significant similarity to the hinge protein of complex III, suggesting that these two polypeptides may be involved in identical functions. This complex I subunit is coded for by a single gene. Applying restriction-fragment-length-polymorphism mapping, we located the gene on the right side of the centromere in linkage group I, linked to the lys-4 locus.
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PMID:The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) from Neurospora crassa: cDNA cloning and chromosomal mapping of the gene. 809 9

It has been suggested that the two acidic regions around residue 70 and residue 170 in yeast cytochrome c1, a subunit of ubiquinol-cytochrome c reductase (complex III), interact with cytochrome c in the electron transfer reaction and that the QCR6 protein, the acidic subunit of yeast complex III, enhances this interaction. In order to determine the roles of the acidic regions of cytochrome c1 more precisely, we introduced several mutations in the two acidic regions and examined their effects on the ability of modified cytochrome c1 to complement the respiration deficiency of yeast cells lacking only cytochrome c1 or both cytochrome c1 and the QCR6 protein. The mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) was still functional in the cytochrome c1-deficient strain. Mutant cytochrome c1 with the deletion of the second acidic region (delta 168-179) caused a decrease in the complementing ability, but this is probably due to failure in its proteolytic maturation and/or correct assembly into complex III. Mutant cytochrome c1 with altered charge distribution in the acidic regions (Asp170Asp171-->Asn170Asn171 or Asp170Asp171-->Asn170Lys171) made the cytochrome c1-deficient cells respiration-competent. On the other hand, mutant cytochrome c1 with the deletion of the first acidic region (delta 68-80) or altered charge distribution in the second region (Asp170Asp171-->Asn170Lys171) did not restore the respiration deficiency of the cells lacking not only cytochrome c1 but also the QCR6 protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acidic regions of cytochrome c1 are essential for ubiquinol-cytochrome c reductase activity in yeast cells lacking the acidic QCR6 protein. 813 52

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase, EC 1.1.2.3) are connected by a hinge peptide. Kinetics experiments [White, P., Manson, F. D. C., Brunt, C. E., Chapman, S. K., & Reid, GA. (1993) Biochem. J. 291, 89-94] have illustrated the importance for efficient interdomain electron transfer of maintaining the structural integrity of the hinge. To probe the role of the hinge in a more subtle manner, we have constructed a mutant enzyme, H delta 3, which has a three amino acid deletion in the hinge region. Intra- and inter-protein electron transfer within H delta 3 flavocytochrome b2 and the H delta 3:cytochrome c redox complex was investigated by steady-state and stopped-flow kinetics analysis. The H delta 3 mutant enzyme remains a good L-lactate dehydrogenase, as is evident from steady-state experiments with ferricyanide as electron acceptor (40% less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (15% less active than wild-type enzyme). The global effect of the deletion is to lower the enzyme's effectiveness as a cytochrome c reductase. This property of the H delta 3 enzyme is manifested at two electron-transfer steps on the catalytic cycle of flavocytochrome b2. First, the rate of heme reduction has fallen 5-fold in H delta 3 compared with the wild-type enzyme (from 445 to 91 s-1), due to poor interdomain electron transfer from flavin to heme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the interdomain hinge of flavocytochrome b2 in intra- and inter-protein electron transfer. 817 86


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