The Interaction of the Rieske Iron-Sulfur Protein with Occupants of the Qo-site of the bc 1 Complex, Probed by Electron Spin Echo Envelope Modulation

Abstract

The bifurcated reaction at the Q o -site of the bc 1 complex provides the mechanistic basis of the proton pumping activity through which the complex conserves redox energy in the proton gradient. Structural information about the binding of quinone at the site is lacking, because the site is vacant in crystals of the native complexes. We now report the first structural characterization of the interaction of the native quinone occupant with the Rieske iron-sulfur protein in the bc 1 complex of Rhodobacter sphaeroides, using high resolution EPR. We have compared the binding configuration in the presence of quinone with the known structures for the complex with stigmatellin and myxothiazol. We have shown by using EPR and orientation-selective electron spin echo envelope modulation (ESEEM) measurements of the iron-sulfur protein that when quinone is present in the site, the isotropic hyperfine constant of one of the N ␦ atoms of a liganding histidine of the [2Fe-2S] cluster is similar to that observed when stigmatellin is present and different from the configuration in the presence of myxothiazol. The spectra also show complementary differences in nitrogen quadrupole splittings in some orientations. We suggest that the EPR characteristics, the ESEEM spectra, and the hyperfine couplings reflect a similar interaction between the iron-sulfur protein and the quinone or stigmatellin and that the N ␦ involved is that of a histidine (equivalent to His-161 in the chicken mitochondrial complex) that forms both a ligand to the cluster and a hydrogen bond with a carbonyl oxygen atom of the Q o -site occupant.The cytochrome (cyt) 1 bc 1 complex family plays an essential role in the energy metabolism of the biosphere (1, 2), providing the central enzymes in the electron transfer chains of all the major pathways. In bacteria, and their mitochondrial and chloroplasts descendants, the complex catalyzes oxidation of quinol and reduction of a diffusible small protein (cyt c or c 2 in ␣-proteobacteria) through a modified Q-cycle (3-6).Interest in the bc 1 complex has been heightened by the recent availability of structures and the new insights into functional aspects that these have encouraged (2, 7-12). However, with the structures has come the realization that the static models do not provide all the information needed to understand mechanism at a deeper level. The reactions of greatest interest are the proton-coupled electron transfers at the quinone-processing sites. In particular, oxidation of ubiquinol at the Q o -site of the complex is the key reaction of the Q-cycle, providing the bifurcation of electron transfer between two different acceptor chains that allows coupling to proton transfer. The Q o -site of the mitochondrial complex, and its mechanism, are also of central importance to the physiology of cellular damage and aging, because it is a major site for generation of reactive oxygen species.To understand the mechanism, we need detailed information about the local reaction environment, including protein structure, hydroge...

Engineering a Three-cysteine, One-histidine Ligand Environment into a New Hyperthermophilic Archaeal Rieske-type [2Fe-2S] Ferredoxin from Sulfolobus solfataricus

“…The major differences in the RR spectra of ARF and sulredoxin are found in the 240 -400 cm Ϫ1 region; interestingly the overall RR features of oxidized ARF are more similar to those reported for the low potential Rieske-type cluster in bacterial phthalate dioxygenase (17,20), which is in line with our recent iron K-edge XAS analysis showing the structural similarity in the coordination environment of the Rieske-type clusters in ARF and bacterial anthranilate dioxygenase (27) (Table I). 6 As an initial step toward understanding the RR spectral difference of the wild-type ARF and sulredoxin, we prepared the uniformly 15 N-labeled wild-type ARF and sulredoxin using 15 NH 4 Cl as a sole nitrogen source in the growth media of E. coli. Successful incorporation of the Rieske-type cluster was detected for both proteins when the cells were grown at 30°C in the modified M9 minimal salt medium containing 15 NH 4 Cl (see "Experimental Procedures").…”

“…The nucleotide sequence of the resultant vector, pTrc99AARF, was confirmed for both strands. The pTrc99AARF vector was transformed into the host strain, E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene), and the transformants were grown for 16 h at 30°C in modified M9 salt medium supplemented with 4.5 g/liter glucose, 1 g/liter 15 Purification of each recombinant holoprotein having a hexahistidine tag at the N terminus was performed as described previously for the S. tokodaii recombinant SdhC iron-sulfur protein (39) except that the heat treatment step (at 65°C for 20 -30 min) was omitted for three variants, C42S, C61S, and H44C. When required, the recombinant holoprotein was further purified by Sephadex G-75 gel filtration column chromatography (Amersham Biosciences).…”

“…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)(3)(4)(5)(12)(13)(14)(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).…”

Functional Insensitivity of the Cytochrome b6f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein

“…Thus, it is inferred that a minimum length of 7 residues in the ISP hinge region is required for optimum binding of the ISP at the Q p site in the b 6 f complex. The decrease in activity and the loss of Q p site inhibitor sensitivity in the 2-residue truncation mutants may be explained by the requirement for H-bonding of quinol and stigmatellin to the His 129 (equivalent to His 161 in bovine) ligand of the [2Fe-2S] cluster for efficient electron transfer from quinol to ISP and inhibitor binding (12,15,48). A truncation of the hinge region would not allow close approach of the [2Fe-2S] cluster at the Q p site and would thus impair the formation of this H-bond.…”

tRNA-modifying MiaE protein fromSalmonella typhimuriumis a nonheme diiron monooxygenase

“…The latter was analyzed by HYSCORE spectroscopy to further characterize the coordination sphere of the diiron center. One of the main advantages of this two-dimensional pulsed EPR technique resides in its ability to distinguish three types of nuclei: the strongly ( ͉ a N ͉ /2 Ͼ N ) and weakly ( ͉ a N ͉ /2 Ͻ N ) coupled ones and the ''distant'' nuclei, which are characterized by very low hyperfine couplings (26,27). In the latter case, the corresponding peaks lie on the diagonal of the (ϩ, ϩ) quadrant, whereas the strongly and weakly coupled nuclei appear in the (Ϫ,ϩ) and the (ϩ, ϩ) quadrants, respectively (28).…”