Tyrosine 147 of Cytochrome b Is Required for Efficient Electron Transfer at the Ubihydroquinone Oxidase Site (Q0) of the Cytochrome bc1 Complex

A. Sami Sariba, University of Pennsylvania Perelman School of Medicine
Huangen Ding, University of Pennsylvania Perelman School of Medicine
P. Leslie Dutton, University of Pennsylvania Perelman School of Medicine
Fevzi Daldal, University of Pennsylvania Perelman School of Medicine


In Rhodobacter capsulatus, tyrosine (Y) 147 is a highly conserved residue of the cyt b subunit of the bc1 complex. It is located in the vicinity of residues altered in spontaneous inhibitor resistant mutants that affect the ubihydroquinone oxidase (Q0) site of this enzyme. In this work, Y147 was substituted with phenylalanine (F), valine (V), serine (S), and alanine (A) using site-directed mutagenesis in an effort to investigate its specific role in the Q0 site. Of the four mutants obtained, Y147S and Y147A exhibited very low ubihydroquinone xyt c reductase activities and were unable to support photosynthetic growth (Ps) while Y147F and Y147V were Ps+. In all mutants, no changes in the redox midpoint potentials (Em7) of the cyt bu and cyt bi, the occupancy of the Q0 site by Q/QH2, and the flash-induced reverse electron transfer kinetics from Qi to cyt bu were observed. On the other hand, rates of electron transfer from Q0 to cyt bu were mildly reduced (2-3-fold) in Y147F and V but dramatically decreased (about 20-fold) in Y147A and S, localizing the defect to the Q0 site. Thus, Y147A and S are members of a novel class of Q0 site mutants that affect the Q0 site catalysis without perturbing the accessibility or binding of the substrate. Additional insight to the role of Y147 on ubihydroquinone oxidation was gained by analyzing the Ps+ revertants of these mutants. Two pseudorevertants contained a second mutation [isoleucine (I) or valine (V)] at the highly conserved M154 position, six residues away from Y147. These hydrophobic second-site substitutions restored electron transfer rates from Q0 to cyt bu to near-wild type levels and conferred resistance to the Q0 site inhibitor, myxothiazol. Considering that Ml54V mutation alone has no effect on the Q0 site, the compensatory effects of the M154V or M1541 substitutions are only needed when position 147 is occupied with a small amino acid side chain such as A or S. These spatial interactions between the positions 147 and 154 suggest that efficient electron transfer at the Q0 site of the bc1 complex requires either bulky side chains at position 147 or hydrophobic side chains at position 154 when the former position is occupied with small residues. Thus, the overall packing and hydrophobicity of the Q0 site appear crucial for maximum catalytic activity. © 1995, American Chemical Society. All rights reserved.