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

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 bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 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 bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 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.

The two [4Fe-4S] clusters in Chromatium vinosum ferredoxin have largely different reduction potentials. Structural origin and functional consequences.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.

Alteration of the midpoint potential and catalytic activity of the rieske iron-sulfur protein by changes of amino acids forming hydrogen bonds to the iron-sulfur cluster.

The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (Sgamma) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme. Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to Sgamma of Cys-159 lowers the midpoint potential by 65 mV. Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly. The activity of the bc1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.

Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE-2S] cluster in Escherichia coli.

The Rieske protein II (Schmidt et al., 1996, FEBS Lett. 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E. coli cells. The full length protein was strictly bound to the E. coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.

Comparison of the "Rieske" [2Fe-2S] center in the bc1 complex and in bacterial dioxygenases by circular dichroism spectroscopy and cyclic voltammetry.

Two different types of "Rieske" [2Fe-2S] clusters have been observed in proteins, one in the bc complexes of the respiratory chain and the other in bacterial dioxygenases. We have compared the circular dichroic (CD) spectra and redox properties of the water soluble fragment of the Rieske center of the bovine heart mitochondrial bc1 complex (ISF) and of the ferredoxin from benzene dioxygenase in Pseudomonas putida ML2 (FDBED). Spinach ferredoxin was also measured for comparison. The redox potential of both proteins could be determined in solution by cyclic voltammetry (CV) and by CD-monitored spectroelectrochemistry using a specially constructed optically transparent thin layer (OTTLE) cell. Whereas the redox potential of the ISF (+312 +/- 5 mV at pH 7.0) depended both on the pH above pH 7 and on the ionic strength, the redox potential of the FDBED (-155 +/- 5 mV at pH 7.0) was observed to be independent of pH and ionic strength. The ISF showed a marked dependence of its redox potential on temperature, while the FDBED showed no temperature dependence. The entropy of the redox reaction delta S degrees rc was calculated as -88 +/- 11 J K-1 mol-1 for the bc1 Rieske center and approximately 0 J K-1 mol-1 for the FdBED. The CD spectra of Rieske type clusters are significantly different from those of plant type [2Fe-2S] ferredoxins. A strong negative CD band is present at 20 000 cm-1 (500 nm) in all reduced Rieske clusters. The possible assignment of this band is discussed as arising from the highest energy magnetically allowed d --> d transition (dz2 --> dxz) of the FeII site. If so, this band is highly indicative of the distortion of the ligand field of the FeII site.