Molecular Control of a Bimodal Distribution of Quinone-Analogue Inhibitor Binding Sites in the Cytochrome b6f Complex

The 3.0–3.1 Å X-ray structures of the cytochrome b6f complex from Mastigocladus laminosus and Chlamydomonas reinhardtii obtained in the presence of the p-side quinone-analogue inhibitor tridecyl-stigmatellin (TDS) are very similar. A difference occurs in the p-side binding position of TDS. In C. reinhardtii, TDS binds in the ring-in mode, as previously found for stigmatellin in X-ray structures of the cytochrome bc1 complex. In this mode, the H-bonding chromone ring moiety of the TDS bound in the Qp niche is proximal to the ISP [2Fe–2S] cluster, and its 13 carbon tail extends through a portal to the large inter-monomer quinone-exchange cavity. However, in M. laminosus, TDS binds in an oppositely oriented ring-out mode, with the tail inserted toward the Qp niche through the portal and the ring caught in the quinone-exchange cavity that is 20 Å away from the [2Fe–2S] cluster. Site-directed mutagenesis of residues that might determine TDS binding was performed with the related transformable cyanobacterium Synechococcus sp. PCC 7002. The following changes in the sensitivity of electron transport activity to TDS and stigmatellin were observed: (a) little effect of mutation L193A in cytochrome b6, which is proximal to the chromone of the ring-out TDS; (b) almost complete loss of sensitivity by mutation L111A in the ISP cluster binding region, which is close to the chromone of the ring-in TDS; (c) a ten and 60-fold increase associated with the mutation L81F in subunit IV. It was inferred that only the ring-in binding mode, in which the ring interacts with residues near the ISP, is inhibitory, and that residue 81 of subunit IV, which resides at the immediate entrance to the Qp niche, controls the relative binding affinity of inhibitor at the two different binding sites.