Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe‐2S] cluster of the cytochrome bc₁ complex in Paracoccus denitrificans

Abstract

Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster.In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc 1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 Oγ-S-1 and Tyr165 Oη-Cys139 Sγ [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567Ϫ579]. They are conserved in all known Rieske iron-sulfur proteins from bc 1 complexes including that from P. denitrificans.Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain.The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc 1 complex isolated from the parental strain was 145 s Ϫ1 , the turnover numbers for the mutants S157A and Y159F were 10 s Ϫ1 and 52 s Ϫ1 , respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential E m of the Rieske cluster at pH 6 and 5°C was ϩ360 mV for the bc 1 complex from the parental strain ; the values in the mutant complexes were ϩ316 mV (Y159F) and ϩ265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.Keywords : Rieske protein; cytochrome bc 1 complex ; Paracoccus denitrificans ; redox potential ; sitedirected mutagenesis.The ubihydroquinone-cytochrome c oxidoreductase is a key and c1 and the Rieske iron-sulfur protein (Yang and Trumpower, 1986; Payne and Trumpower, 1987). The iron-sulfur protein was component of photosynthetic and respiratory electron-transport chains in many organisms. The complex is embedded in the in-first found in bovine heart mitochondrial membranes by Rieske et al. (1964) and has later been identified as the oxidation factor ner mitochondrial membrane of eukaryotes or in the plasma membrane of many bacteria. It catalyzes the reduction of cyto-of the cytochrome bc 1 complex (Trumpower and Edwards, 1979). In contrast to [2Fe-2S] clusters, e.g. of plant type ferrechrome c by ubihydroquinone and translocates protons across the membrane by the Q-cycle mechanism.doxins, Rieske [2Fe-2S] clusters are coordinated by two cysteine and two histidine residues (Gurbiel et al., 1989(Gurbiel et al., , 1991; Britt et In contrast to mitochondrial cytochrome bc 1 complexes containing 10 or 11 subunits, bacterial cytochrome bc 1 complexes al., 1991). In the recently solved X-ray structure of the watersoluble fragment (ISF) of the Rieske iron-sulfur protein f...

Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex

“…The Y156W strain showed a slightly increased affinity of the substrate, QH 2 , which was also predicted in the K QH2 estimation. The K m value of the S154A strain was close to that of wild type, in contrast with the previous report from Schroter et al (25) but in line with the relatively small effects on K QH2 for this strain. The estimated V max values of all the strains were similar to the V opt values measured in pH titration, as discussed below.…”

“…The [2Fe-2S] cluster of the ISP has a net charge of 0 or Ϫ1 for the oxidized and reduced forms, respectively. From sequence difference, and the wide range of potentials in other [2Fe-2S] clusters, it is clear that the redox properties of the cluster are affected by protein environment (23)(24)(25)(26)(27). When sequences from bacterial species with different substrates are aligned, differences at particular positions can be correlated with the difference in the redox properties of the cluster and used to guide selection of sites for mutagenesis (15, 24 -26, 28).…”

“…These two residues thus contribute substantially to the electronegativity responsible for the high potential (E m ϳ300 mV in mitochondria and ␣-proteobacteria (30)) and low pK a values (30 -32). Strains mutated at these and several other residue positions were produced in the bc 1 complex from R. sphaeroides (24,28), Paracoccus denitrificans (25), and in yeast (26). From the variation in rate with driving force, each of these laboratories suggested that the first electron transfer must control the overall rate of QH 2 oxidation.…”

Asymmetric Binding of Stigmatellin to the Dimeric Paracoccus denitrificans bc1 Complex

“…All inhibitors as well as DBH 2 were quantified by UV-spectroscopy (10) using reported extinction coefficients (11,12). 1 ComplexThe Y159F mutation was introduced with the Altered Sites system (Promega, Madison, WI) as described previously (13). The fbc operon encoding the wild-type or the mutated P. denitrificans bc 1 complex was cloned into the HindIII/SacI sites of the vector pRI2 (14).…”

The Small Subunit AroB of Arsenite Oxidase

“…The strong sequence diversity in this group (both from wild type and from mutated proteins) and the number of available crystal structures now allow for a reliable analysis of the individual effects of residues surrounding the cluster. This kind of analysis has already been carried out on the subgroup of the Rieske-cyt b proteins (38,41,42,54). The Aro subgroup of Rieske proteins has not yet been examined in this way.…”