Tyrosine 147 of cytochrome b is required for efficient electron transfer at the ubihydroquinone oxidase site (Qo) of the cytochrome bc1 complex.
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 (Qo) 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 Qo site. Of the four mutants obtained, Y147S and Y147A exhibited very low ubihydroquinone:cyt 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 bH and cyt bL, the occupancy of the Qo site by Q/QH2, and the flash-induced reverse electron transfer kinetics from Qi to cyt bH were observed. On the other hand, rates of electron transfer from Qo to cyt bH 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 Qo site. Thus, Y147A and S are members of a novel class of Qo site mutants that affect the Qo 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biology, Johnson Research Foundation, University of Pennsylvania, Philadelphia 19104.
A S
AS
Saribaş
H
H
Ding
P L
PL
Dutton
F
F
Daldal
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D008961:Models, Structural; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D016354:Rhodobacter capsulatus / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D014443:Tyrosine; D014450:Electron Transport Complex III