We heterologously overproduced a hyperthermostable archaeal low potential (E m ‫؍‬ ؊62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys 42 and Cys 61 ) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Riesketype ferredoxin appear to be inequivalent as indicated by much higher stability of the His 64 Proteins containing Rieske-type [2Fe-2S] clusters are widespread in nature from hyperthermophilic Archaea and Bacteria to Eukarya and play critical electron transfer roles in various pathways such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (1-4). In contrast to regular plant-and vertebrate-type ferredoxins having complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron-sulfur core with the S ␥ atom of each of the two cysteine residues coordinated to one iron site and the N ␦ atom of each of the two histidine residues coordinated to the other iron site. This asymmetric ligation results in some unique redox and spectroscopic properties (for reviews, see Refs. 1 and 3-5). This cluster coordination was firmly established by recent x-ray crystal structures of several different Rieske-type protein domains (6 -11).Two different types of Rieske clusters are observed in proteins. One type displays higher reduction potentials (E m ) 1 of approximately ϩ150 to ϩ490 mV and occurs in proton-translocating respiratory complexes (cytochrome bc 1 /b 6 f complexes and their archaeal homologs without c-type cytochromes), being involved in not only electron transfer but also substrate binding and oxidation at the quinol-oxidizing Q o site (2-5, 12-15). The other type displays lower E m values of approximately Ϫ150 to Ϫ50 mV and has been found in a diverse group of bacterial multicomponent terminal oxygenases and soluble Rieske-type ferredoxins (1, 3, 8, 9, 16 -27). However, none of the latter class has been characterized in detail from any archaeal species.We recently found that the genomic DNA sequence of the thermoacidophilic archaeon Sulfolobus solfataricus strain P-1 (DSM 1616T) encodes an archaeal homolog of bacterial small Rieske-type ferredoxins with no consensus disulfide signature (DDBJ accession number AB047031 (27)). This arf gene was found by homology search against the deduced amino acid sequence of Sulfolobus tokodaii sulredoxin, a water-soluble homolog of a high potential Rieske protein (E m,low pH ϳ ϩ190 mV) with a consensus disulfide linkage (DDBJ accession number AB023295) 2 (28 -30) (Fig. 1). Subsequent cloning and heterologous overexpression in Escherichia coli of this S. solfataricus arf gene encoding the archaeal Rieske-type ferredoxin (ARF) (27) have provided an opportunity to define the influence of surrounding amino acid residues on the electronic and s...

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Proteins of the Rieske and Rieske-type family contain a [2Fe-2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome bc-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. (14)N ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8-10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.

The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.

The weakly coupled 15N atoms around a reduced Rieske [2Fe-2S] cluster of the uniformly 15N-labeled, hyperthermostable archaeal Rieske protein appear to produce readily observable cross-peaks in the HYSCORE spectra, with the well-resolved couplings of 0.3-0.4 MHz for the Nepsilon and 1.1 MHz for the peptide backbone nitrogens, in addition to the contributions from the coordinated Ndelta atoms. These features can be used for structure-mechanism studies of the biological redox protein system involving the weakly coupled nitrogens in coupled electron-proton transfer reactions.

CW EPR spectra of reduced [2Fe-2S](Cys)3(His)1 clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly 15N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 Nδ ligand with hyperfine coupling 15a=8 MHz. The 14N and 15N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies ~2.8 and 2.4 MHz (N2), and the other ~4.0 and 3.5 MHz (N1), but did not show any of the larger splitting ~1.1–1.4 MHz previously seen with Rieske proteins. Further analysis with partially 15N(3)-His-labeled protein indicates that His87 Nε cross-peaks produce resolved features (N2) in the 14N spectrum but contribute much less than weakly-coupled peptide nitrogen species to the (++) quadrant in the 15N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S] 2؉ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms.Respiratory complex II (succinate:quinone oxidoreductase) is an iron-sulfur (FeS) 1 flavoprotein complex that serves as the sole membrane-bound component of the oxidative tricarboxylic acid cycle as well as one of the most important primary dehydrogenases at the electron entry site of the aerobic respiratory chain for a variety of aerobic organisms from archaea to bacteria to eukarya (1-4). In general, mesophilic bacterial and eukaryal enzymes consist of three to four different subunits. The largest flavoprotein subunit (SdhA) contains the dicarboxylate active site at a covalently linked FAD via 8␣-[N(3)-histidyl] linkage, and the second largest FeS protein subunit (SdhB) contains a high potential [2Fe-2S] cluster (Center S-1), a low potential [4Fe-4S] cluster (Center S-2), and a high potential [3Fe-4S] cluster (Center S-3) (1, 2, 4) (see Fig. 1, A and B). The membrane anchor subunits bind quinones involved in the electron transfer reactions of the enzyme, and in some cases, they contain one or two protohemes IX as prosthetic groups (1, 2, 4).Recently, the crystal structures of closely related enzymes, fumarate reductase (Frd) complexes of Escherichia coli and Wolinella succinogenes, which are key terminal enzymes in the bacterial anaerobic respiratory chains and catalyze fumarate reduction, were solved at 3.3-and 2.2-Å resolution, respectively (5, 6). These structures revealed the presence of a covalently bound FAD and three FeS clusters in a nearly linear cofactor arrangement with the common sequence, FAD-[2Fe-2S]-[4Fe-4S]-[3Fe-4S] (4 -6) (see Fig. 1B). In contrast, marked variation was noted between the membrane anchor subunits involved in the electron transfer reactions, which bind two menaquinone molecules in the E. coli complex (5) and two protoheme centers in the W. succinogenes complex (6), both arranged vertically relative to the cytoplasmic membranes.The aerobic r...

The hyperfine couplings for strongly and weakly coupled 15N nuclei around a reduced Rieske [2Fe-2S] center of uniformly 15N-labeled, hyperthermostable archaeal Rieske protein at pH 13.3 were determined by hyperfine sublevel correlation (HYSCORE) spectroscopy and compared with those at physiological pH. Significant changes in the hyperfine couplings of the terminal histidine Ndelta ligands and Nepsilon nuclei were observed between them, which can be explained by not only the redistribution of the unpaired electron spin density over the ligands but also the difference in the mixed-valence state of the fully deprotonated, reduced cluster. These quantitative data can be used in theoretical analysis for the selection of an appropriate model of the mixed-valence state of the reduced Rieske center at very alkaline pH.