Decoupling mutations in the D-channel of the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides suggest that a continuous hydrogen-bonded chain of waters is essential for proton pumping.
The aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides utilizes two proton-input channels to provide all the protons for chemistry (water formation) and proton pumping. The D-channel is responsible for the uptake of all pumped protons, four protons per O(2). Several substitutions of either N139 or N207, near the entrance of the D-channel, were previously reported to decouple the proton pump from oxidase activity. In this work, the characteristics of additional mutations in this region of the protein (N139, N207, N121, and S142) are determined to elucidate the mechanism of decoupling. With the exception of the substitution of a large, hydrophobic residue (N139L), all the mutations of N139 resulted in an enzyme with high oxidase activity but with a severely diminished proton pumping stoichiometry. Whereas N207D was previously shown to be decoupled, N207A and N207T exhibit nearly wild-type behavior. The new data display a pattern. Small, nonionizable substitutions of N139 or N121 result in decoupling of the proton pump but maintain high turnover rates. These residues are directly hydrogen bonded to two water molecules (Water6574 and Water6584) that are part of the single-file chain of water molecules within the D-channel leading to E286 at the top of the channel. The data suggest that the integrity of this water chain within the D-channel is critical for rapid proton transfer. The mechanism of decoupling is most likely due to the slowing of the rate of proton delivery below a threshold that is required for protonation of the putative proton loading site. Protons delivered outside this time window are delivered to the active site where they are consumed in the formation of water. The rate of proton delivery required to protonate the pump site must be significantly faster than the rate of delivery of protons to the catalytic site. For this reason, mutations can result in decoupling of the proton pump without slowing the catalytic turnover by the enzyme.
Zhu
Jiapeng
J
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
Han
Huazhi
H
Pawate
Ashtamurthy
A
Gennis
Robert B
RB
D001665:Binding Sites / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D009154:Mutation; D017494:Proton Pumps / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D014867:Water / Q000378:metabolism
D017494:Proton Pumps; D011522:Protons; D014867:Water; D003576:Electron Transport Complex IV