Hyperfine Sublevel Correlation Spectroscopy Studies of Iron−Sulfur Cluster in Rieske Protein from Green Sulfur Bacterium Chlorobaculum tepidum Hiroki Nagashima, † Hiraku Kishimoto, ‡ Risa Mutoh, §,∥ Naotaka Terashima, † Hirozo Oh-oka, ‡ Genji Kurisu, ‡,§ and Hiroyuki Mino* ,† † Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan ‡ Department of Biological Sciences, Graduate School of Science, Osaka University, Suita, Osaka 560-0043, Japan § Institute for Protein Research, Osaka University, Toyonaka, Osaka 565-0871, Japan * S Supporting Information ABSTRACT: The magnetic properties of the Rieske protein purified from Chlorobaculum tepidum were investigated using electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy (HYSCORE). The g-values of the Fe 2 S 2 center were g x = 1.81, g y = 1.90, and g z = 2.03. Four classes of nitrogen signals were obtained by HYSCORE. Nitrogens 1 and 2 had relatively strong magnetic hyperfine couplings and were assigned as the nitrogen directly ligated to Fe. Nitrogens 3 and 4 had relatively weak magnetic hyperfine couplings and were assigned as the other nitrogen of the His ligands and peptide nitrogen connected to the sulfur atom via hydrogen bonding, respectively. The anisotropy of nitrogen 3 reflects the different spin density distributions on the His ligands, which influences the electron transfer to quinone. 1. INTRODUCTION Cytochrome (cyt) bc complexes are crucial energy-transducing machinery in respiratory and photosynthetic electron-transport chains. They conduct characteristic quinol oxidation reactions that couple electron transport with proton translocation across membranes, resulting in the production of an electrochemical proton gradient or proton motive force essential for adenosine 5′-triphosphate synthesis. These reactions are now well understood as the Q-cycle mechanism. 1 Recently, genome- wide analyses have shown that the minimal functional and mechanistic unit of the Q-cycle mechanism consists of Rieske iron−sulfur protein (ISP) and cyt b. 2 This catalytic core evolved into various complexes by incorporating structurally unrelated proteins. For example, cyt bc 1 and b 6 f complexes have incorporated cyt c 1 and f, respectively, as secondary electron carriers. In phototrophic green sulfur bacteria (or Chlorobia- ceae), the petC gene coding for c-type cyt is absent in the transcriptional unit of the petCB genes for Rieske ISP and cyt b, indicating that its cyt bc complex would be of the Rieske/cyt b type. 3,4 Moreover, phylogenetic analyses have also suggested that cyt b in green sulfur bacteria splits into cyt b 6 and subunit IV before its divergence to cyt b 6 -type complexes in heliobacteria (cyt b 6 cc) and cyanobacteria (cyt b 6 f). 5 The Rieske/cyt b unit utilizes two categorized pool quinones, that is, low- and high-potential quinones, to produce the proton motive force across membranes. 6 The low-potential menaqui- none (MK, E m = ∼−70 mV) is considered to be the ancestral type of quinone. Vast ranges of prokaryotes, including green sulfur bacteria and heliobacteria, which inhabit mostly anaerobic environments, oxidize menaquinol at the Q o site to induce the bifurcated electron transfer reactions. On the other hand, almost all proteobacteria (and mitochondria), including phototrophic purple bacteria, cyanobacteria (and chloroplasts), and hyperthermophilic aerobic archaebacteria, which adapted to the modern oxygenic atmosphere, possess the high-potential quinones (E m = ∼100 mV): ubiquinone (proteobacteria), plastoquinone (PQ) (cyanobacteria), and caldariellaquinone (Sulfolobus spp.). From the viewpoint of evolutionary processes in chemiosmotic energy-converting systems, it would have been critical for MK-type cyt bc complexes to commence utilization of higher potential quinones in the transition from anaerobic to aerobic conditions, induced by the action of oxygenic photosynthesis. 2 Without any upshift of the redox potentials of cofactors in this enzyme, especially those of the Rieske cluster, higher potential quinones were very poor electron donors for the retrieval of electrochemical energy. In fact, the Rieske/cyt b complex in green sulfur bacteria is positioned just before the divergence to a PQ-type cyt b 6 f Received: December 25, 2016 Revised: March 1, 2017 Published: March 2, 2017 Article pubs.acs.org/JPCB © XXXX American Chemical Society A DOI: 10.1021/acs.jpcb.6b12968 J. Phys. Chem. B XXXX, XXX, XXX−XXX