PROTON COUPLING IN THE CYTOCHROME-P-450 SPIN AND REDOX EQUILIBRIA

Ferric bacterial cytochrome P-450 is known to exist as a mixture of high-spin (S = 5/2) and low-spin (S = 1 /2) configurations of the heme iron d-shell electrons. This equilibrium between the two forms of P-450 has been shown to control both substrate affinity and the observed redox potential. Room temperature binding of a solvent-exchangeable proton to high-spin, substrate-free P-450 occurs with pK = 6.5, while camphor association shifts this equilibrium to a pK of 5.8. No H+ ligation to the low-spin forms of P-450 is observed. Proton coupling is also observed in the reduction of camphor-bound, high-spin ferric P-450. Analysis of these structures by a four-state, free-energy coupling model yields pKs of 5.8 and 8.0 for H+ binding respectively to Fe3+ and Fe2+ P-450 and inherent electrode potentials of -106 and -238 mV for the oxidation/reduction of protonated and unprotonated protein. Analysis of the free-energy interaction diagram yields the standard state potential E°' = -173 mV at pH 7 for camphor-bound material. Proton modulation of the putidaredoxin-cytochrome P-450 electron transfer in a stabilized dienzyme complex is quantitated by stopped-flow spectrophotometry. Protonation of the multiprotein complex is seen to occur on cytochrome reduction-redoxin oxidation with pK = 5.8, indicating that, at pH 7, there is little equilibration with solvent protons. The possibility of concerted H+/e- transfer during P-450-catalyzed, mixed function oxidation is discussed. © 1979, American Chemical Society. All rights reserved.