The ferric spin-state equilibrium and relaxation rate of cytochrome P-450 has been examined with temperature jump spectroscopy using a number of camphor analogues known to induce different mixed spin states in the substrate-bound complexes [Gould, P., Gelb, M., & Sligar, S.G. (1981) J. Biol. Chem. 256, 6686]. All temperature-induced spectral changes were monophasic, and the spin-state relaxation rate reached a limiting value at high substrate concentrations. The ferric spin equilibrium constant, is defined in terms of the rate constants kx and via = [P-450(HS)]/[P-450(LS)] where HS and LS represent high-spin (S = 5/2) and low-spin (S = l/2) ferric iron, respectively, and the spectrally observed spin-state relaxation rate by kohsd= kx+ k-X. A strong correlation between the fraction of high-spin species and the rate constant, is observed. For a 3 °C temperature jump (from 10 to 13 °C), the 23% high-spin tetramethylcyclohexanone complex = 45 ± 20 is characterized by a ferric spin relaxation rate of = 1990 s-1, while the rates for the d-fenchone (41% high spin, Kd= 42 ± 10 and camphoroquinone (75% high spin, Kd= 15 ± 5 complexes are 1430 and 346 respectively. The transition rates from high- to low-spin cytochrome with these substrates are 1380, 675, and 75 respectively, suggesting that the decrease in which correlates with the increasing fraction of high-spin species present at equilibrium, is due to a restricted access and recombination rate of the water axial ligand with the ferric iron.
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