Heterogeneous Rieske Proteins in the Cytochrome b6f Complex of Synechocystis PCC6803?

Dirk Schneider,Sven Skrzypczak,Stefan Anemüller,Christian L Schmidt,Andreas Seidler,Matthias Rögner

doi:10.1074/jbc.m104076200

Dirk Schneider, Sven Skrzypczak + Show 4 more

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https://doi.org/10.1074/jbc.m104076200

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Abstract

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b(6)f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b(6)f complexes in Synechocystis and suggests still to be established alternative electron transport routes.

Highlights

The functional core of cytochrome bc complexes consists of three subunits, which are a b-type cytochrome, a c-type cytochrome, and the Rieske iron-sulfur protein [1]
It was shown that substitutions of single amino acids within or around these boxes affect the midpoint potential of the Rieske proteins [7] and that this midpoint potential can be predicted to some extent from the amino acid sequence
In organisms utilizing menaquinone, one or both of the lateral residues Ser and Tyr are replaced by nonhydroxylated amino acids resulting in a lowered midpoint potential of the Rieske protein

Summary

Introduction

The functional core of cytochrome bc complexes consists of three subunits, which are a b-type cytochrome, a c-type cytochrome, and the Rieske iron-sulfur protein [1]. It is likely that this unusual coordination causes the high midpoint potential of about ϩ100 to ϩ400 mV Besides these coordinating amino acids there are two additional cysteine residues, which form a disulfide bridge close to the ironsulfur cluster. Proteins with the sequence CPCHGSXY of the C-terminal ironsulfur cluster-binding site (Box II) oxidize high potential quinones like ubi- and plastoquinone, and these proteins have a midpoint potential between ϩ250 and ϩ375 mV. The previously described overexpression of the Synechocystis Rieske protein PetC1 yielded predominantly a non-native form clustered in inclusion bodies [10]. Both studies agree that a small fraction of the overexpressed protein can be found in E. coli membranes, without incorporated iron-sulfur cluster.

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