A cytochrome C electron transfer switch modulated by heme ligation and isomerization of a peptidyl‐prolyl bond

Abstract

Intermolecular electron transfer (ET) between hexaamineruthenium(II), a(6) Ru(2+) , and a K73H/K79A variant of iso-1-cytochrome c, iso-1-Cytc, is used to study conformational ET switches mediated by His73-heme ligation and cis to trans isomerization of the Ile75-Pro76 peptidyl-prolyl bond of iso-1-Cytc. The biomolecular rate constant for ET to the native state of K73H/K79A iso-1-Cytc is ∼270 mM(-1) s(-1) near neutral pH. The unimolecular conformational ET switches due to His73-heme ligation and the Ile75-Pro76 peptidyl-prolyl bond gate ET at rate constants of 5 to 10 s(-1) and 0.05 to 0.06 s(-1) . Thus, at 1 mM a(6) Ru(2+) , these conformational ET switches slow electron transfer by about 50- and 5000-fold, respectively. The conformational ET switches are populated between pH 5 and 7, providing a means of modulating ET in this redox protein over several orders of magnitude by simply changing pH. The conformationally-gated ET measurements are analyzed in the context of previous pH jump measurements on the His73-heme alkaline transition of K73H/K79A iso-1-Cytc. The ability to obtain microscopic rate constants with conformationally-gated ET measurements has allowed more precise determination of the pK(a) s of the three ionizable groups that mediate population of the His73-heme ET switch. We have also been able to show that the ionizable group with a pK(a) near 9 stabilizes the His73-heme conformer relative to the native state of iso-1-Cytc and that contrary to the conclusions from our pH jump studies, this ionization does not strongly affect the rate of the Ile75-Pro76 peptidyl-prolyl isomerization.