Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the (2Fe-2S) center

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (Mason and Cammack 1992; Trumpower and Gennis 1994; Link 1999; Berry et al. 2000). Their distinct biological function is in part associated with the cluster redox potential (Em), for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed (Iwata et al. 1996; Colbert et al. 2000; Hunsicker-Wang et al. 2003). Despite this fundamental importance, evaluation of the contribution of structure to function for each hydrogen bond in the immediate cluster environment is often difficult to address experimentally in many iron-sulfur proteins, because several hydrogen bonds are contributed by the peptide backbone (Iwata et al. 1996; Colbert et al. 2000; Hunsicker-Wang et al. 2003; Iwasaki et al. 2004b). In the cytochrome (cyt) bc1/b6f family, a Rieske protein contributes to the high-potential electron transfer chain involved in the bifurcated mechanism at the quinol-oxidizing Qo site, catalyzing a crucial step in the coupled electron–proton transfer that is linked to generation of a transmembrane electrochemical proton gradient (Berry et al. 2000; Crofts 2004). All of its Sb/t atoms are involved in a complex hydrogen bonded network (through which the cluster is linked to the surrounding polypeptide chain), which includes contributions from the side-chain hydroxyl groups of highly conserved Ser-154 (to Sb) and Tyr-156 (to St of Cys-129) (in Rhodobacter sphaeroides numbering [Guergova-Kuras et al. 2000]; equivalent to Ser-163 and Tyr-165, respectively, in bovine numbering [Iwata et al. 1996]) (Fig. ​(Fig.1A).1A). This peculiarity has provided an opportunity to explore the influence of these hydrogen bonds on the cluster Em of the cyt bc1-associated Rieske proteins by site-directed mutagenesis (Denke et al. 1998; Schroter et al. 1998; Guergova-Kuras et al. 2000). Elimination of each hydroxyl group lowers the cluster Em, changes the EPR spectrum of the reduced Rieske [2Fe-2S] center, and decreases the rate of ubiquinol oxidation by cyt bc1 complex (Denke et al. 1998; Schroter et al. 1998; Guergova-Kuras et al. 2000). In at least one case, it also influences the pKa,ox value, which was attributed to the Nɛ of the histidyl ligands of the cluster (Guergova-Kuras et al. 2000). Although these studies have provided the basis for understanding its particular mechanistic importance, the nature of each hydrogen bond and its contribution to the immediate cluster environment have not been investigated in detail. Figure 1. The structures of the Rieske [2Fe-2S] cluster site in R. sphaeroides ISP (A) (taken from 1rie.pdb but in R. sphaeroides numbering [Guergova-Kuras et al. 2000]) and S. tokodaii SDX (B) (Uchiyama et al. 2004; T. Iwasaki, T. Uchiyama, A. Kounosu, and T. Kumasaka, ... We report here a comparative study of variations in resonance Raman (RR) spectra of a repertoire of bacterial and archaeal high-potential Rieske proteins with different hydrogen-bond networks. These have been varied by site-directed mutagenesis in three well-characterized mutant proteins of the Rieske cluster binding domain solubilized by proteolytic cleavage from the hydrophobic N-terminal tail (ISP) of R. sphaeroides cyt bc1 complex (Em, acid pH = +315 mV; pKa,ox1, 7.6 ± 0.1) (Fig. ​(Fig.1A;1A; Guergova-Kuras et al. 2000; Zu et al. 2003) and two mutant proteins of the high-potential, archaeal sulredoxin (SDX) from Sulfolobus tokodaii strain 7 (Em, acid pH = +188 mV; pKa,ox1 of the visible CD transition [corresponding to pKa,ox2 of the Em], 8.4 ± 0.2) (Iwasaki et al. 1995, 1996, 2004a; Kounosu et al. 2004; Uchiyama et al. 2004) with weak homology with the regular cyt bc1-associated Rieske proteins (DDBJ-EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB023295","term_id":"11527554","term_text":"AB023295"}}AB023295) (Fig. 1B). In contrast with the wild-type ISP, there is no hydrogen-bonding interaction between the Sb/t atoms and side-chain groups in the wild-type SDX because of the presence of Ala-66 and Phe-68 in place of Ser-154 and Tyr-156, respectively, as confirmed by the preliminary crystal structure of the wild-type SDX being refined at 2.0 A resolution (Fig. ​(Fig.1B;1B; Uchiyama et al. 2004; T. Iwasaki, T. Uchiyama, A. Kounosu, and T. Kumasaka, unpubl.).