Conformational equilibriums accompanying the electron transfer between cytochrome c (P551) and azurin from Pseudomonas aeruginosa

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Abstract
The redox reaction between cytochrome c (Cyt c) (P-551) and the blue Cu protein azurin, both from P. aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve 3 equilibria. The fast relaxation time (0.4 < .tau. < 8 ms) was ascribed to the electron exchange step. The slow relaxation time (.tau. .simeq. 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). Because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the 2 slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (.tau. .simeq. 0.1 ms) would be zero. At 25.degree. C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 .times. 106 and 7.8 .times. 106 M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at .apprx. 104 s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35.degree. C), i.e. .DELTA.Htot = 0. This zero enthalpy change was compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were 2-3.times. higher than the values published for various low molecular weight reagents in their electron exchange with Cu proteins, yet the rate of exchange between Cyt c (P551) and azurin was hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer space sphere reaction, of which the rate could be approximated by the Marcus theory.

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