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.

Covalent complex between yeast cytochrome c and beef heart cytochrome c oxidase which is active in electron transfer

Biochemistry ◽  
1981 ◽  
Vol 20 (24) ◽  
pp. 7046-7053 ◽  
Author(s):  
Stephen D. Fuller ◽  
Victor M. Darley-Usmar ◽  
Roderick A. Capaldi
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Beef Heart ◽  
Covalent Complex

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.

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

1992 ◽  
Vol 70 (5) ◽  
pp. 301-308 ◽  
Author(s):  
M. Crinson ◽  
P. Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Reduction Rate ◽  
Artificial Substrates ◽  
Intramolecular Electron Transfer ◽  
State Reduction ◽  
Hydrogen Donors ◽  
Slow Turnover

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT – cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (≈70% of the total with resting and ≈85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.Key words: cytochrome c oxidase, cytochrome a, respiration, cyanide, stopped flow.

Control of proteoliposomal cytochrome c oxidase: the partial reactions

1990 ◽  
Vol 68 (9) ◽  
pp. 1135-1141 ◽  
Author(s):  
Peter Nicholls
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Membrane Potential ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Redox State ◽  
Ph Gradient ◽  
Transfer Model ◽  
State Reduction

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith–Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by ΔpH and by ΔΨ can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.Key words: proteoliposomes, cytochrome c, cytochrome oxidase, membrane potential, pH gradient, cytochrome a, electron transfer.

Cytochrome c reduction by cysteine plus copper: a pseudosubstrate system for cytochrome c oxidase

1980 ◽  
Vol 58 (6) ◽  
pp. 499-503 ◽  
Author(s):  
Bruce C. Hill ◽  
Peter Nicholls
Keyword(s):  
Electron Transfer ◽  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Electrode ◽  
Horse Heart Cytochrome ◽  
Ferricytochrome C ◽  
Cytochrome C Reduction ◽  
Low Ionic Strength ◽  
Horse Heart Cytochrome C

Cysteine alone reduces horse heart cytochrome c very slowly [Formula: see text] with a rate constant virtually identical in high and low ionic strength buffers. Copper catalyzes this reaction increasing the rate by a factor of 105 in 50 mM phosphate and by a factor of 106 in 10 mM Tris buffers. When ferricytochrome c and cysteine are mixed in an oxygen electrode a "burst" of oxygen uptake is seen, the decline in which parallels the reduction of cytochrome c. When cytochrome c oxidase is added to such a mixture two routes of electron transfer to oxygen exist: enzymatic and ferricytochrome c dependent nonenzymatic. Both processes are sensitive to cyanide, but azide inhibits only the authentic cytochrome c oxidase catalyzed process and BCS the ferricytochrome c stimulated reaction.

Effects of subunit V antibodies on the topology of the subunit and the activity of beef heart cytochrome-c oxidase

1988 ◽  
Vol 66 (11) ◽  
pp. 1210-1217 ◽  
Author(s):  
Jo A. Freedman ◽  
Bryan Leece ◽  
Christopher E. Cooper ◽  
Peter Nicholls ◽  
Samuel H. P. Chan
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Redox State ◽  
Quantitative Comparison ◽  
Beef Heart ◽  
Specific Antibodies ◽  
Antibody Preparations

Redox-sensitive epitopes on subunit V of beef heart cytochrome-c oxidase were demonstrated previously using polyclonal subunit-specific antibodies raised in rabbits. The antibodies only slightly inhibited electron transfer, and the accessibility of their epitopes depended on the presence of a membrane and on the redox state of the oxidase. The present paper describes additional preparations of antibodies raised against subunit V. These antibodies have an even higher subunit specificity, they are more than three times as inhibitory against electron transfer, and their binding does not require a membrane. Moreover, the redox-sensitive nature of their binding to detergent-dispersed oxidase is sensitive to the method of its isolation. We discuss inferences that can be drawn from a detailed quantitative comparison of the interactions of the two antibody preparations with the antigen in different environments. The techniques used in the comparison can be used to examine other perturbants of the oxidase as to their effects on specific segments of the enzyme.

scholarly journals Cloning and sequencing of the cDNA for a 13th different subunit (IHQ) of beef heart cytochrome c oxidase

1989 ◽  
Vol 264 (28) ◽  
pp. 16858-16861
Author(s):  
R Lightowlers ◽  
S Takamiya ◽  
R Wessling ◽  
M Lindorfer ◽  
R A Capaldi
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Beef Heart ◽  
Cloning And Sequencing
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