Cross-Linked Electron Transfer Complex between Cytochrome c2 and the Photosynthetic Reaction Center of Rhodobacter sphaeroides

Abstract

Electron donation from the soluble cytochrome (cyt) c2 to the photooxidized primary donor, P+, of reaction centers isolated from Rhodobacter 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 c2. In 80% of the reaction centers, P+ generated by a laser flash is reduced by covalently bound cyt c2. Kinetics of P+ reduction show (i) a fast phase with a half-life of 0.7 micros similar to that observed for electron transfer in the noncovalent proximal complex and (ii) a slow phase (t1/2 = 60 micros) that is attributed to a cyt c2 bound less favorably for electron transfer. Its relationship with similar kinetic phases attributed to a distal conformation of the complex in previous studies is discussed. Both kinetic phases are slightly accelerated upon addition of glycerol. Upon addition of reduced soluble cyt c2 to the cross-linked complex the kinetics of both phases are not affected. The kinetics of P+ reduction following the second flash (20 ms after the first) show that a complex is formed between soluble cyt c2 and the cross-linked complex, in which electron transfer takes place in the millisecond time domain. Cross-linked cyt c2 in complexes which give rise to the two kinetic phases of P+ reduction shows almost pH-independent midpoint redox potentials between pH 6 and 9.5. This behavior is at variance with that of free cyt c2, the midpoint potential of which is affected by at least two protonable groups within this pH range. The cross-linked RC-cyt c2 complex allowed study of the effects of temperature on the electron transfer reaction without a possible disturbance by dissociation of the complex. In the 250-300 K range, Arrhenius behavior is observed showing activation energies of 11.7 and 8.0 kJ/mol for the faster and the slower kinetic phases, respectively, which are remarkably lower than the activation energy of 20.5 kJ/mol for the fast P+ reduction by soluble cyt c2 [Venturoli, G., Mallardi, A., & Mathis, P. (1993) Biochemistry 32, 13245-13253]. Between 250 and 230 K, a fall-off in amplitude is observed for both kinetic phases indicating that intracomplex electron transfer is blocked at low temperatures.