Refine
Year of publication
- 1998 (2)
Document Type
- Article (2)
Language
- English (2)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Institute
- Medizin (2)
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.
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.