Photooxidation of mitochondrial cytochrome c by isolated bacterial reaction centers: Evidence for tight-binding and diffusional pathways

Abstract

The binding of horse heart mitochondrial cytochrome c to isolated reaction centers from Rhodopseudomonas sphaeroides is described. The kinetics of photooxidation of cytochrome c following a short actinic flash is compared to the expected binding state of the cytochrome at various concentrations and at different ionic strengths. At low ionic strength a very tight binding site (KD≦10(-8) M) is apparent which is nonfunctional with respect to electron donation to the bound reaction center. This tightly bound cytochrome can react with another reaction center in a diffusion limited, second order process. A weaker binding site (KD≃0.3 · 10(-6) M) is also boserved which is associated with rapid, first order electron transfer from cytochrome to reaction center. Both binding processes are weakened in the presence of salt and there is no detectable binding in 100 mM NaCl. Under such conditions cytochrome oxidation is entirely a diffusional, second order process. However, analysis of the flash intensity dependence of the extent of cytochrome oxidation, by the method of van Grondelle (van Grondelle, R. (1978) Ph.D. Thesis, State University, Leiden) indicated that the cytochrome was not freely mobile even in 100 mM NaCl, at least in the sense that reduced cytochrome only slowly dissociates from unactivated reaction centers. An overall kinetic/equilibrium scheme for cytochrome c binding and photooxidation by reaction centers is presented. This is very similar to that described earlier for cytochrome c2 (Overfield, R.E., Wraight, C.A. and DeVault, D. (1979) FEBS Lett. 105, 137-142), but the tight binding site and associated diffusion controlled oxidation is unique to cytochrome c.

Solution structure of a biological bimolecular electron transfer complex: characterization of the photosynthetic reaction center-cytochrome c2 protein complex by small angle neutron scattering

“…in which two types of RC-cytochrome c 2 complexes are formed that are capable of either fast, k et , or conformationally gated, k b , electron transfer (Overfield & Wraight, 1986;Moser & Dutton, 1988). With saturating cytochrome concentrations, these electron transfer studies show strongly bi-phasic kinetics.…”

Interaction between Cytochrome c2 and Reaction Centers from Purple Bacteria

“…For the two-state model, electron transfer has a fast phase, with a time constant of ~1 ps, due to bound cytochrome and a slow component due to the reaction of cytochrome in solution (Rosen et al, 1979). Other workers have proposed that the data are more consistent with a three-state model in which there are two fast components due to binding of cytochrome c2 in both a distal and a proximal state (Dutton & Prince, 1978;Moser & Dutton, 1988;Overfield & Wraight, 1986). It has been suggested that the difference in these results is due to variations in nonintrinsic properties of the RC that are dependent upon the isolation process (Tiede et al, 1993).…”

Role of specific lysine residues in binding cytochrome c2 to the Rhodobacter sphaeroides reaction center in optimal orientation for rapid electron transfer

“…The positively charged horse cytochrome c binds about 10-fold more strongly to the proximal site than the negatively charged cytochrome c2 (K¿ = 0.3 vs 3.0 µ ), but the rate for the fast phase of the reaction is slower (r1/l2 f This work was supported by NIH Grants GM20488 and RR07101. 0006-2960/89/0428-6970S01.50/0 = 2 vs 0.6 ps) (Rosen et al, 1979; Overfield & Wraight, 1986).…”