Protein-protein interaction and electron transfer: Case of cytochrome f and cytochrome c6

We used photo-induced reaction as a tool to look to explore dynamics of the protein-protein interaction between cytochrome f and cytochrome c6 from C. reinhardtii. In our studies of electron transfer between 3Zncyt c6 and cyt f(III) we discovered two distinct intracomplex processes corresponding to electron transfer within diprotein complexes termed persistent and transient. The overall reaction remained biphasic and the rate constants for both processes were invariant to the change of ionic strength within the studied interval. The docking of these proteins is due to strong hydrophobic interaction slightly augmented by weak electrostatic attraction. We used covalent cross-linking and size-exclusion chromatography to capture redox-inactive dimers of cyt f(III). Analysis of the temperature and viscosity effects on the overall reaction between cyt c6 and cyt f revealed that reaction within persistent complex is true electron transfer, but mechanism of reaction within transient complex switches from coupled to gated at the temperature of ca. 303°K. This is the first case, to our knowledge, that same net reaction (oxido-reduction in this case) within one metalloprotein pair occurs simultaneously as true and gated, or true and coupled electron transfer. We propose the equation that adequately treats adiabatic and nonadiabatic features of coupled mechanism. We show that the difference between three regimes, true, coupled, and gated, is not semantic but real. Furthermore, these regimes can be distinguished experimentally. The simultaneous occurrence of all three mechanisms within the same metalloprotein pair allowed us to estimate the energetics of protein interfacial rearrangement that modulates mechanism of electron-transfer reaction. Brownian dynamics calculations support dynamic picture that we inferred from the kinetics studies. Comparison of cyt c6 and plastocyanin in their reaction with cyt f revealed that the identical function and seemingly very similar physico-chemical properties of the two proteins are not a guarantee that the common reaction will follow a common mechanism. We derived from literature data and our own studies that the energetics of configurational interconversion of the complex, rather than the initial configuration, will determine the mechanism of the electron-transfer reactions.