Aromatic amino acids in the Rieske iron–sulfur protein do not form an obligatory conduit for electron transfer from the iron–sulfur cluster to the heme of cytochrome c1 in the cytochrome bc1 complex

Abstract

We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron-sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron-sulfur protein of the yeast cytochrome bc1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron-sulfur proteins, except for those containing a W152L and a W176L-F177L double mutation, for which the activity was approximately 45%. The crystal structures of the bc1 complex indicate that F177 and H184 are at the surface of the iron-sulfur protein near the surface of cytochrome c1, but not directly in a linear pathway between the iron-sulfur cluster and the c1 heme. The pre-steady-state rates of reduction of cytochromes b and c1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70-85% of the wild-type rates. There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L-F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.

Analysis of the Nucleus-Encoded and Chloroplast-Targeted Rieske Protein by Classic and Site-Directed Mutagenesis of Chlamydomonas

“…Both petC-W163R and petC-W163A mutants showed little or no phototrophy. Analysis of the petC-W163R and petC-Y87D mutants indicates that certain aromatic residues are necessary for Rieske protein stability and assembly, as are specific aromatic residues of the yeast bc 1 protein (Snyder et al, 1999). Furthermore, we find that perhaps as a consequence of structural changes, electron transfer is considerably slowed in these mutants that assemble a portion of the mutant Rieske proteins into only partially functional b 6 f complexes.…”

Elimination of the Disulfide Bridge in the Rieske Iron−Sulfur Protein Allows Assembly of the [2Fe-2S] Cluster into the Rieske Protein but Damages the Ubiquinol Oxidation Site in the Cytochrome bc1 Complex

“…The strains were grown in 20 L carboys at room temperature on synthetic dropout medium containing 0.7% YNB, 0.14% amino acid mix lacking uracil, and 2% dextrose with aeration. Cells were harvested by centrifugation and broken in a Waring Blender in liquid nitrogen (12).…”

Mechanism on Two-Electron Oxidation of Ubiquinol at the Qp Site in Cytochrome bc1 Complex: B3LYP Study with Broken Symmetry

“…The Cbc 1 catalyzes twoelectron oxidation of ubiquinol (UQH 2 ) to yield ubiquinone (UQ) at the Q p site in the proton-motive Q cycle. [7][8][9][10][11][12] The Q p site of 1KYO (PDB code) 5 consists of the Rieske iron-sulfur [2Fe-2S] cluster of ISP and Glu272 of cytochrome b, as shown in Figure 1. The inhibitor stigmatellin interacts simultaneously with both Glu272 and His181 coordinating to the iron of the Rieske iron-sulfur [2Fe-2S] cluster by the hydrogen bonds.…”

“…Although the X-ray structures where UQH 2 or UQ is docked between the [2Fe-2S] cluster and Glu272 instead of the inhibitors are not observed yet, it has been proposed that the oxidation proceeds by docking the UQH 2 between His181 and Glu272. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] There is common consensus in the oxidation of UQH 2 that the reaction proceeds in bifurcated electron-transfer (ET) mechanism with one electron reducing the [2Fe-2S] cluster while the other electron reduces a cyt b heme (b L ). 25,26 After the [2Fe-2S] cluster receives one electron, the [2Fe-2S] cluster moves to cytochrme c 1 (c 1 position) to deliver the electron to cytochrome c through the cytochrme c 1 .…”