Negative Cooperativity in the Reduction of Excitonically Interacting B-Hemes of the Cytochrome B6F Complex

Abstract

Cytochrome b6f and bc1 (bc) complexes, which provide the central charge transfer complex in respiratory and photosynthetic electron transport chains, are symmetric dimeric structures.1 A trans-membrane electron transfer pathway between hemes bp and bn2 exists in each monomer. Based on inter-heme distances this intra-monomer pathway (bp-bn) is preferred over an inter-monomer cross-over pathway between hemes bp1. Cross-over has been reported,3,4 although the branching ratio is not known. Previous studies on ferredoxin-dependent reduction of b6f by NADPH showed only half of the b-heme reduced in thylakoid membranes,5 and biphasic reduction with isolated complex.6 It was inferred that the rapid reduction occurred through intra-monomer electron transfer and that the split circular dichroism spectrum7,8 (node of the CD spectrum in the Soret band coincides with the heme absorbance maximum), implies that the hemes bp and bn interact excitonically in the monomeric unit. Here, monophasic chemical (dithionite) reduction of monomeric b6f complex, characterized by size-exclusion chromatography, proceeds 10-20 times more rapidly than in the dimeric complex. The reduced monomer showed full amplitude of the Soret band split CD spectrum. These measurements are consistent with “half-sites reactivity” observed for b-heme reduction in the yeast bc1 complex,9,10 in which reduction of one monomer is associated with a reduced rate of electron transfer to the second monomer of the bc1 dimer. The mechanism for this negative cooperativity could arise from a larger reorganization energy11 for inter-monomer electron transfer, or constraints imposed by the Rieske protein in the dimer. NIH-GM38323.1Cramer et al. 2011; 2Yamashita et al. 2007; 3Lanciano et al. 2010; 4Swierczek et al. 2010; 5Furbacher et al. 1989; 6Hasan et al., 2011; 7Palmer and Degli-Esposti 1994; 8Schoepp et al. 2000; 9Covian and Trumpower, 2008; 10Castellani et al. 2010; 11Marcus and Sutin 1985.