Cytochrome c Oxidase: Biphasic Kinetics Result from Incomplete Reduction of Cytochrome a by Cytochrome c Bound to the High-Affinity Site

Biochemistry ◽  
1995 ◽  
Vol 34 (31) ◽  
pp. 10000-10008 ◽  
Author(s):  
Jaime Ortega-Lopez ◽  
Neal C. Robinson
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Biphasic Kinetics ◽  
High Affinity ◽  
High Affinity Site

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

1980 ◽  
Vol 58 (10) ◽  
pp. 969-977 ◽  
Author(s):  
P. Nicholls ◽  
V. Hildebrandt ◽  
B. C. Hill ◽  
F. Nicholls ◽  
J. M. Wrigglesworth
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
The Other ◽  
Enzyme Preparations ◽  
Apparent Affinity ◽  
High Affinity ◽  
High Affinity Site ◽  
Membrane Bound ◽  
Enzyme Molecules ◽  
Low Ionic Strength

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 μM > Kd′ > 0.2 μM) and readily reversible and the other of high affinity (0.01 μM > Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s−1 (30 °C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites are occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c – cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.

scholarly journals Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking

1996 ◽  
Vol 315 (3) ◽  
pp. 909-916 ◽  
Author(s):  
Francesco MALATESTA ◽  
Giovanni ANTONINI ◽  
Flavia NICOLETTI ◽  
Alessandro GIUFFRÈ ◽  
Emilio D'ITRI ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Beef Heart ◽  
State Reduction ◽  
High Affinity ◽  
Disulphide Bond Formation ◽  
High Affinity Site ◽  
Low Ionic Strength ◽  
Covalent Complex

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046–7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) Km and Vmax values obtained from the non-linear Eadie–Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4

1991 ◽  
Vol 11 (10) ◽  
pp. 4934-4942
Author(s):  
J C Schneider ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Biogenesis ◽  
Binding Sites ◽  
Single Gene ◽  
Regulatory Region ◽  
Gene Encoding ◽  
High Affinity ◽  
High Affinity Site ◽  
Cytochrome C1 ◽  
The Difference

Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.

scholarly journals An unusual cytochrome o′-type cytochrome c oxidase in a Bacillus cereus cytochrome a 3 mutant has a very high affinity for oxygen

Microbiology ◽  
1999 ◽  
Vol 145 (7) ◽  
pp. 1563-1573 ◽  
Author(s):  
Martha L. Contreras ◽  
J. Edgardo Escamilla ◽  
I. Patricia Del Arenal ◽  
J. R. Dávila ◽  
Rita D'mello ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Bacillus Cereus ◽  
Cytochrome C Oxidase ◽  
High Affinity ◽  
Cytochrome O ◽  
Very High

scholarly journals Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase

1982 ◽  
Vol 202 (2) ◽  
pp. 527-534 ◽  
Author(s):  
R J Diggens ◽  
C I Ragan
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxidase Activity ◽  
Maximal Activity ◽  
Complex Iii ◽  
Protein Ratio ◽  
Quinol Oxidase ◽  
Cytochrome C Reductase ◽  
Ubiquinol Oxidase ◽  
High Affinity

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.

Role of phospholipid fatty acids on the kinetics of high and low affinity sites of cytochrome c oxidase

1986 ◽  
Vol 64 (11) ◽  
pp. 1195-1210 ◽  
Author(s):  
A. Trivedi ◽  
D. J. Fantin ◽  
E. Reno Tustanoff
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Membrane Fluidity ◽  
Mitochondrial Membrane ◽  
Polar Head Group ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
High Affinity

The nature of the interactions between cytochrome c oxidase and the phospholipids in mitochondrial membranes has been investigated by varying the nature of the fatty acyl components of Saccharomyces cerevisiae. A double fatty acid yeast mutant, FAI-4C, grown in combinations of unsaturated (oleic, linoleic, linolenic, and eicosenoic) and saturated (lauric and palmitic) fatty acids, was employed to modify mitochondrial membranes. The supplemented fatty acids constituted a unique combination of different acyl chain lengths with varying degrees of unsaturation which were subsequently incorporated into mitochondrial phospholipids. Phosphatidylethanolamine and cardiolipin, the predominant phospholipids of the inner mitochondrial membrane, were characterized by their high levels of supplemented unsaturated fatty acids. Increasing the chain length or the degree of unsaturation of mitochondrial membrane phospholipids had no effect on altering the nature of the phospholipid polar head group but did result in a profound change on the specific activity of cytochrome c oxidase. When studied under conditions of different ionic strengths and pHs the enzyme's activity, as documented by Eadie–Hofstee plots, showed biphasic kinetics. The kinetic parameters for the low affinity reaction were greatly influenced by the changes in the membrane fatty acids and only marginal effects were noted at the high affinity reaction site. The discontinuities in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, monitored at increasing temperatures, suggested that changes in membrane fluidity were conditioned by alterations in mitochondrial membrane fatty acid constituents. These results indicate that the lipid changes affecting the low affinity binding site of cytochrome c oxidase may be the result of lipid–protein interactions which lead to enzyme conformational changes or may be due to gross changes in membrane fluidity. It may, therefore, follow that this enzyme site may be embedded in or be juxtaposed to the outer surface of the inner mitochondrial membrane bilayer in contrast to the high affinity site which has been shown to be significantly above the membrane plane.

scholarly journals Inhibition of membrane-bound cytochrome c oxidase by zinc ions: High-affinity Zn2+ -binding site at the P-side of the membrane

FEBS Letters ◽  
2008 ◽  
Vol 582 (30) ◽  
pp. 4158-4162 ◽  
Author(s):  
Tatiana V. Vygodina ◽  
Wiolanta Zakirzianova ◽  
Alexander A. Konstantinov
Keyword(s):  
Cytochrome C ◽  
Binding Site ◽  
Cytochrome C Oxidase ◽  
Zinc Ions ◽  
High Affinity ◽  
Membrane Bound

scholarly journals Regulation of the yeast CYT1 gene encoding cytochrome c1 by HAP1 and HAP2/3/4.

1991 ◽  
Vol 11 (10) ◽  
pp. 4934-4942 ◽  
Author(s):  
J C Schneider ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Biogenesis ◽  
Binding Sites ◽  
Single Gene ◽  
Regulatory Region ◽  
Gene Encoding ◽  
High Affinity ◽  
High Affinity Site ◽  
Cytochrome C1 ◽  
The Difference

Mitochondrial biogenesis requires the coordinate induction of hundreds of genes that reside in the nucleus. We describe here a study of the regulation of the nuclear-encoded cytochrome c1 of the b-c1 complex. Unlike cytochrome c, which is encoded by two genes, CYC1 and CYC7, c1 is encoded by a single gene, CYT1. The regulatory region of the CYT1 promoter contains binding sites for the HAP1 and HAP2/3/4 transactivators that regulate CYC1. The binding of HAP1 to the CYT1 element was studied in detail and found to differ in two important respects from binding to the CYC1 element. First, while CYC1 contains two sites that bind HAP1 cooperatively, CYT1 has a single high-affinity site. Second, while the CYT1 site and the stronger HAP1-binding site of CYC1 share a large block of homology, the HAP1 footprints at these sites are offset by several nucleotides. We discuss how these differences in HAP1 binding might relate to the difference in the biology of cytochrome c and cytochrome c1.

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