CROSS-LINKED ELECTRON-TRANSFER COMPLEX BETWEEN CYTOCHROME C(2) AND THE PHOTOSYNTHETIC REACTION-CENTER OF RHODOBACTER-SPHAEROIDES

Electron donation from the soluble cytochrome (cyt) c(2) to the photoo xidized primary donor, P+, of reaction centers isolated from Rhodobact er sphaeroides was studied by using chemical zero-length cross-linking . This cross-linking stabilizes a 1:1 covalent complex between subunit M of the reaction center and cyt c(2). In 80% of the reaction centers , P+ generated by a laser flash is reduced by covalently bound cyt c(2 ). Kinetics of P+ reduction show (i) a fast phase with a half-life of 0.7 mu s similar to that observed for electron transfer in the noncova lent proximal complex and (ii) a slow phase (t(1/2) 60 mu s) that is a ttributed to a cyt c(2) bound less favorably for electron transfer. It s relationship with similar kinetic phases attributed to a distal conf ormation of the complex in previous studies is discussed. Both kinetic phases are slightly accelerated upon addition of glycerol, Upon addit ion of reduced soluble cyt c(2) to the cross-linked complex the kineti cs of both phases are not affected. The kinetics of P+ reduction follo wing the second flash (20 ms after the first) show that a complex is f ormed between soluble cyt c(2) and the cross-linked complex, in which electron transfer takes place in the millisecond time domain. Cross-li nked cyt c(2) in complexes which give rise to the two kinetic phases o f P+ reduction shows almost pH-independent midpoint redox potentials b etween pH 6 and 9.5. This behavior is at variance with that of free cy t c(2), the midpoint potential of which is affected by at least two pr otonable groups within this pH range. The cross-linked RC-cyt c(2) com plex allowed study of the effects of temperature on the electron trans fer reaction without a possible disturbance by dissociation of the com plex. In the 250-300 K range, Arrhenius behavior is observed showing a ctivation energies of 11.7 and 8.0 kJ/mol for the faster and the slowe r kinetic phases, respectively, which are remarkably lower than the ac tivation energy of 20.5 kJ/mol for the fast P+ reduction by soluble cy t c(2) [Venturoli, G., Mallardi, A., & Mathis, P. (1993) Biochemistry 32, 13245-13253]. Between 250 and 230 K, a fall-off in amplitude is ob served for both kinetic phases indicating that intracomplex electron t ransfer is blocked at low temperatures.