Proton transfer in cytochrome bo3 ubiquinol oxidase of Escherichia coli: second-site mutations in subunit I that restore proton pumping in the mutant Asp135-->Asn.
The ubiquinol oxidase, cytochrome bo3, of Escherichia coli is a member of the respiratory heme-copper oxidase family and conserves energy from the reduction of dioxygen to water by translocation of protons across the bacterial membrane. Mutation of an aspartic acid residue (Asp135) to asparagine in subunit I of this enzyme was previously found to impair proton translocation [Thomas et al. (1993) Biochemistry 32, 10923-10928]. This residue is located in an interhelical "loop" between transmembranous helices II and III, which contains six well-conserved residues (Asn124, Pro128, Gly132, Asp135, Pro139, and Asn142). Site-directed mutagenesis was performed to study the function of this entire domain. Nonconservative mutations of Asn124 and Asn142 also resulted in a loss of proton translocation, whereas their conservative substitution to glutamine had no effect. Mutations in eight other positions within this domain did not affect proton translocation. Introduction of an acidic group at positions 139 or 142, but not at eight other tested positions, restored proton pumping in the Asp135-->Asn mutated protein. These results suggest that the C-terminal part of the domain may be alpha-helical and that the entire "loop" plays an important structural and functional role as part of an input channel of the proton translocation machinery.
Garcia-Horsman
J A
JA
School of Chemical Sciences, University of Illinois at Urbana-Champaign 61801, USA.
Puustinen
A
A
Gennis
R B
RB
Wikström
M
M
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017494:Proton Pumps / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017494:Proton Pumps; D001224:Aspartic Acid; D001216:Asparagine; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D008239:Lysine