Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump.
Cytochrome oxidase catalyzes the reduction of O2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all of the pumped protons enter the enzyme through the D channel and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing oxidase activity. N139 is one of three asparagines near the entrance of the D channel, where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pKa of E286, a functionally critical residue that is located 20 A away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pKa of E286. This finding reinforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286, which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.
Han
Dan
D
Department of Biochemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Namslauer
Andreas
A
Pawate
Ashtamurthy
A
Morgan
Joel E
JE
Nagy
Stanislav
S
Vakkasoglu
Ahmet S
AS
Brzezinski
Peter
P
Gennis
Robert B
RB
D001216:Asparagine / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009154:Mutation; D017494:Proton Pumps / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D017494:Proton Pumps; D001224:Aspartic Acid; D001216:Asparagine; D003576:Electron Transport Complex IV