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Potential Ligands to the [2Fe-2S] Rieske Cluster of the Cytochrome bcl Complex of Rhodobacter capsulatus Probed by Site-Directed Mutagenesis+ Edgar Davidson,t Tomoko Ohnishi,s Emmanuel Atta-Asafo-Adjei,lJ and Fevzi Daldal**t Department of Biology, Plant Science Institute, and Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania I91 04 Received November 19, 1991; Revised Manuscript Received January 22, I992 ABSTRACT: The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bel complex or b$complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu,C.-A., Yu,L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R.J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-1 15841. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Q,) site of the bcl complex.

%e ubiquinol-cytochrome c oxidoreductase (bc, complex)' is a key component of photosynthetic and respiratory electron-transport chains in many organisms, and is a site where energy transfer is coupled to ATP synthesis (Dutton, 1986). The subunit composition of this membrane-bound complex

varies with the organism, but irrespective of the source,all have three components with the following functional prosthetic groups: a cytochrome b containing two b hemes, a cytochrome cI containing a c heme, and an ironsulfur protein, commonly referred to as the Rieske ironsulfur (Fe-S) protein, which

This work was supported by NIH Grants GM 38237 (to F.D.) and NSF DMB-8819305 (to T.O.). * Author to whom correspondence should be addressed. *Departmentof Biology, University of Pennsylvania. f Department of Biochemistry and Biophysics, University of Pennsylvania. 11 Present address: Laboratory of Cellular and Molecular Pharmacology, NIEHS, Research Triangle Park, NC 27709.

Abbreviations: bp, base [email protected]);bc, complex, ubiquinokytochrome oxidoreductase; cyt, cytochrome; DBH, 2,3-dimethoxy-5-methyl-6decyl- 1 ,rl-benzoquinone;E,, redox midpoint potential; EPR, electron paramagnetic resonance; MOPS, 3-(N-morpholino)propanesulfonic acid; PMS, phenazine methosulfate; Qo, quinol oxidation site; Qi, quinone reduction site; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ps, photosynthetic growth. c

0006-2960/92/043 1-3342%03.00/0 0 1992 American Chemical Society

Ligands to the Rieske Cluster of Cyt bcl contains a [2Fe-2S] cluster. These subunits form two quinone binding sites, Q, and Qi, where the quinones are oxidized and reduced, respectively [for general reviews, see Cramer and Knaff (1990) and Trumpower (1990)l. The photosynthetic bacterium Rhodobacter capsulatus uses a bc, complex in both photosynthetic and respiratory electron transfer, and its isolated complex contains only the three proteins described above. The structural genes for these proteins, thefbcFBC (petABC) operon, have been cloned and sequenced (Gabellini & Sebald, 1986; Daldal et al., 1987; Davidson & Daldal, 1987). A subsequent combination of molecular genetics (Daldal et al., 1989; Atta-Asafo-Adjei & Daldal, 1991) and biophysical analyses (Robertson et al., 1990) has allowed the identification of regions, and specific amino acid residues, of cytochrome b which are thought to be involved in the oxidation of ubiquinol at the Q, site. The investigation of mutations in these regions has provided a working model for the Q, site (Robertson et al., 1990). In contrast to the wealth of information concerning cytochrome b, relatively little is known about the Rieske protein whose function is to oxidize the QH, at the Q, site and subsequently pass an electron to cytochrome cl. The bulk of the protein, with the [2Fe-2S] cluster, is thought to the exposed in the periplasm, but the precise topology of its membrane attachment has yet to be defined. Most of our knowledge is centered on the [2Fe-2S] cluster itself [see Trumpower (198l)], On the basis of the Mossbauer and resonance Raman spectroscopic features, the Rieske Fe-S protein has been inferred to have a binuclear-type cluster structure [see Fee et al. (1986)l. The distinct electron paramagnetic resonance (EPR) spectrum of the Rieske protein has been well characterized and exhibits a unique spectral line shape [with g values g , , , = 1.80, 1-90,and 2.03, respectively, with an average g value (gav= 1.91) which is lower than that for the ferredoxin (Fd)-type [2Fe-2S] cluster (gav= 1.96)]. Its redox midpoint potential (E,), around +300 mV, is much higher (by -700 mV) than that of typical ferredoxins [see, e.g., Fee et al. (1986)l. The g, feature of the Rieske spectrum (g, = 1.80 in ascorbate-reduced samples) is of particular interest since its line shape and g value are sensitive to the redox state of the Q pool in the membrane, presumably through interaction at the Q, site (Matsuura et al., 1983). Q, site inhibitors alter the E , and EPR line shape of the Rieske cluster, strongly implying that the Rieske protein is part of the Q, site. However, to date no Q, inhibitor-resistant mutation has yet been mapped to the Rieske protein (di Rag0 et al., 1988; Howell & Gilbert, 1988; Daldal et al., 1989). One basic piece of structural information which is not known is the identity of the amino acid ligands to the cluster. The identification of the residues which bind the [2Fe-2S] cluster to the protein is the focus of this work. The Rieske proteins share their characteristic biophysical features, i.e., relatively high E , and EPR line shape, with several bacterial dioxygenases which are involved in degradation of aromatic compounds. These enzymes contain two different species of [2Fe-2S] cluster, one of which is frequently referred to as a “Rieske-type” cluster [e.g., see Fee et al. (1986)l. Blumberg and Peisach (1974) proposed that, for the Rieske type of cluster, at least one of the ligands was less electron-donating than sulfur. Subsequent research has confirmed this suggestion. Studies using a number of biophysical techniques, including electron nuclear double resonance (ENDOR) and Massbauer spectroscopy, have implied that the [2Fe-2S] cluster of dioxygenases contains two sulfur ligands and at least one ni-

Biochemistry, Vol. 31, No. 13, 1992 3343 trogen ligand (Cline et al., 1985). ENDOR and electron spin echo envelope modulation (ESEEM) studies on yeast mitochondrial bcl complex have suggested that the Rieske [2Fe-2S] cluster contained at least one, and probably two, nitrogen ligand (Telser et al., 1987). Extended X-ray absorption fine structure (EXAFS) studies of bovine heart mitochondrial bc, complex also eliminated exclusive sulfur or nitrogen ligations (Powers et al., 1989). The presence of two nitrogen ligands in the Rieske-like [2Fe-2S] cluster of Pseudomonas cepacia phthalate dioxygenase was firmly established by ENDOR spectroscopy (both X- and Q-band) after isolation of this dioxygenase from cells grown on 14N-or 15N-enrichedmedia (Gurbiel et al., 1989). During the course of the experiments reported here, ESEEM (Britt et al., 1991) and ENDOR (Gurbiel et al., 1991) studies using purified bc, complexes provided evidence that two nitrogen and two sulfur atoms are ligands to the [2Fe-2S] cluster of the Rieske protein in various cytochrome bc, and b$complexes. Amino acid sequences of the Rieske protein have been obtained from a number of sources: mitochondrial, chloroplast, and from several species of bacteria [see Hauska et al. (1988)l. The carboxyl terminus of the Rieske protein has been implicated in the binding of its [2Fe-2S] cluster on the basis of proteolytic cleavage (Li et al., 1983). Examination of the available sequence data shows that this region of the protein features two groups of six entirely conserved residues, called here box I (CTHLGC) and box I1 (CPCHGS). Each box contains two cysteines (Cys) and one histidine (His) (Figure l), residues most likely to provide sulfur and nitrogen ligands to the cluster. Comparison of available amino acid sequences has previously proven useful in assigning the His ligands to the two b-type hemes of cytochrome b of the be, complex. When all cytochrome b sequences, including cytochrome bb, from a range of species were compared, it was found that only four of the many histidines were universally conserved (Widger et al., 1984; Saraste et al., 1984). Unfortunately, a comparison of all available Rieske sequences shows that the number of potential ligands available as Cys and His residues exceeds the number needed (Figure 1). The four Cys and two His residues of boxes I and I1 are universally conserved; His 159 (in the R . capsulatus sequence numbering) is not but is sometimes substituted by a Gln (Figure 1) which may also provide a nitrogen ligand (Gatti et al., 1989). When the first Rieske protein sequence was obtained, it was assumed that the [2Fe-2S] cluster was analogous to the [2Fe-2S] cluster of many ferredoxins in being liganded by four Cys residues (Harnisch et al., 1985). Since then, several groups [e.g., see Beckman et al. (1987) and Gatti et al. (1989)l have speculated on the identity of the ligands to the Rieske [2Fe-2S] cluster. However, it is not currently known which of these conserved Cys and His residues are the ligands of the [2Fe-2S] cluster. Our experiments have therefore sought to define the liganding residues by using site-directed mutagenesis to alter all potential ligands. For this purpose, we have obtained 19 substitutions in 7 conserved positions of the Rieske protein of R . capsulatus and analyzed their biochemical and biophysical properties, the results of which are reported here. We have also examined the effects of these mutations on the steady-state stability and biochemical properties of the cytochrome b and cytochrome c1 subunits in membranes; these studies are reported in the following paper (Davidson et al., 1992). MATERIALS AND METHODS Media, Bacterial Strains, and Growth Conditions. The growth of R . capsulatus and Escherichia coli strains on

Davidson et al.

3344 Biochemistry, Vol. 31, No. 13, 1992 I

Table I: Characteristics of R. capsulatus Rieske FeS Protein Mutants mutation box strains QH~cY I MTO-404 wild type 100 C133ARG TGC-CGC 0.0 C133SER TGC-AGC 0.2 H135LEU CAC-CTC ND H135PRO CAC-CCC 0.1 C138ARG TGC-CGC ND C138SER TGC-AGC ND ND C138PHE TGC-TTC



s.c N.c. Mzm. Tbm. P.d B.j


R.r. R.s.



N. Ag.


. ..



159 HIS

“GGTGCACAC-3’; petA-C 138, 5’-ATCGGCAC(GI C)(A/C/T)(N)GCCGAGGTG-3’; petA-(2153, 5’TGGCAGGGGC(T /G / CrGAACCAG- 3’; pet A-C 15 5 , 5’GAGCCGTGlG/CMA/C/T)(NZGCAGGGGCA-3’; petAH 156, 5’-TGCGAGCClG/C)NNGCAGGGGCA-3’; petAH 159, 5’-AATCGTAiG/C)NNCGAGCCGTGG-3’. The merodiploid R . capsulatus strains containing plasmid-borne mutations were constructed essentially as shown in Figure 2 of Atta-Asafo-Adjei and Daldal (1991), except that the 600 bp BstXI-EcoRI (instead of the 450 bp Eco-


10-9 10-8 3 x 10-8 6 X lod

ND 2 x 10-8 0.0

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound ...
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