D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate

The histidine-specific reagent diethyl pyrocarbonate has been used to chemically modify bovine heart cytochrome oxidase. Thirty-two of sixty-seven histidine residues of cytochrome oxidase are accessible to modification by diethyl pyrocarbonate. Effects on the Soret and α bands of the heme spectrum indicate disturbance in the environment of one or both of the heme groups. However, diethyl pyrocarbonate modification does not alter the 830-nm absorbance band, suggesting that the environment of CuA is unchanged. Maximal modification of cytochrome oxidase by diethyl pyrocarbonate results in loss of 85–90% of the steay-state electron transfer activity, which can be reversed by hydroxylamine treatment. However, modification of the first 20 histidines does not alter either activity or the heme spectrum, but only when 32 residues have been modified are the activity and heme spectral changes complete. The steady-state kinetic profile of fully modified oxidase is monophasic; the phase corresponding to tight cytochrome c binding and low turnover is retained, whereas the high turnover phase is abolished. Proteoliposomes incorporated with modified oxidase have a 65% lower respiratory control ratio and 40% lower proton pumping stoichiometry than liposomes containing unmodified oxidase. These results are discussed in terms of a redox-linked proton pumping model for energy coupling via cytochrome oxidase.Key words: cytochrome oxidase, histidine modification, electron transfer, proton pumping, diethyl pyrocarbonate.

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Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1991.tb16162.x ◽

1991 ◽

Vol 199 (3) ◽

pp. 615-622 ◽

Cited By ~ 26

Author(s):

Andre B. P. KUILENBURG ◽

Henk L. DEKKER ◽

Coby BOGERT ◽

Popko NIEBOER ◽

Bob F. GELDER ◽

...

Keyword(s):

Steady State ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Subunit Composition ◽

Kinetic Properties ◽

Oxidase Subunit ◽

Steady State Kinetic ◽

Human Cytochrome ◽

State Kinetic ◽

Human Cytochrome C

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Some spectral and steady-state kinetic properties of Pseudomonas cytochrome oxidase

Biochemical Journal ◽

10.1042/bj1570431 ◽

1976 ◽

Vol 157 (2) ◽

pp. 431-438 ◽

Cited By ~ 44

Author(s):

D Barber ◽

S R Parr ◽

C Greenwood

Keyword(s):

Steady State ◽

Cytochrome Oxidase ◽

Nitrite Reductase ◽

Reductase Activity ◽

Sedimentation Coefficient ◽

Oxidation Products ◽

Sodium Ascorbate ◽

Kinetic Properties ◽

Steady State Kinetic ◽

State Kinetic

Some spectra of Pseudomonas cytochrome oxidase are reported, both for comparison with those of other workers and to illustrate the differences between the ascorbate- and dithionite-reduced forms of the enzyme. A spectrum of the reduced enzyme-CO complex, prepared in the absence of added reductants by incubation under CO, is also included. Ultracentrifugation studies yielded a value for the sedimentation coefficient (s20,w) of 7.5S, and an isoelectric point of pH6.9 was determined by isoelectric focusing. Steady-state kinetic constants of the electron donors, quinol, sodium ascorbate, reduced Pseudomonas azurin and Pseudomonas ferrocytochrome c551 were investigated giving Km values of 30mM, 4mM, 49muM and 5.6muM respectively. The two protein substrates were observed to be subject to product inhibition and the Ki for oxidized Pseudomonas azurin was evaluated at 4.9muM. Steady-state kinetics were also used to investigate the effects of the oxidation products of dithionite on the oxidase and nitrite reductase activities of Pseudomonas cytochrome oxidase. These experiments showed that whereas the oxidase activity was inhibited, the nitrite reductase activity was slightly enhanced.

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