Structure-function on the p-side of the cytochrome b6f complex of oxygenic photosynthesis

Jiusheng Yan, Purdue University

Abstract

The 3.0–3.4 Å X-ray structures of the cytochrome b6f complex from the thermophilic cyanobacterium, M. laminosus and the green alga, C. reinhardtii, in the presence/absence of quinone analogue inhibitors, TDS and DBMIB, revealed 3 interesting features on its p-side: (i) the [2Fe-2S] cluster of iron-sulfur protein (ISP) is too distant from the heme of cytochrome f to allow an efficient electron transfer; (ii) the TDS pharmacophore binds at the active site, the quinol-oxidation (Qp) pocket, in a normal “ring-in” mode in the C. reinhardtii structure, but at a site outside the Qp-pocket in a novel “ring-out” mode in the M. laminosus structure; (iii) DBMIB also binds at an unexpected peripheral site outside the Qp-pocket. In the present studies, the mechanism of electron transfer between the ISP and cytochrome f, and the binding and inhibition mechanism of TDS and DBMIB, were investigated by site-directed mutagenesis and spectrophotometric assays. The following conclusions were reached from these studies: (a) as observed in the mitochondrial cytochrome bc1 complex, the ISP soluble domain is able to move to narrow the distance gap in spinach b6f complex; (b) however, in contrast to the bc1 complex, the function of the b6f complex is not sensitive to changes in length and flexibility of the ISP pivot hinge region, implying a major difference between these two complexes; (c) only the “ring-in” binding mode of TDS is inhibitory; (d) inhibition of electron transport by DBMIB arises from a “structurally unseen” low affinity site within the Qp-pocket, where the binding affinity of DBMIB is greatly increased after a catalytic turnover of the b6 f complex; (e) the statistical distribution of TDS in the “ring-in” and “ring-out” binding modes is determined by a single residue change (L81F in subunit IV), located at the immediate entrance to the Q p-pocket; (f) the observed non-inhibitory binding modes of TDS and DBMIB represent intermediate states during their transport into and out of the Qp-pocket. These studies provided insight into a large scale domain movement of an iron-sulfur protein, and demonstrated pathways and intermediate states for transport of lipophilic quinone/analogue molecules through a membrane protein complex.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Molecular biology|Biophysics|Microbiology

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