Redesign of the proton-pumping machinery of cytochrome c oxidase: proton pumping does not require Glu(I-286).
One of the putative proton-transfer pathways leading from solution toward the binuclear center in many cytochrome c oxidases is the D-pathway, so-called because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-286), has been indicated to play a central role in the proton-pumping machinery of mitochondrial-type enzymes, a role that requires a movement of the side chain between two distinct positions. In the present work we have relocated the glutamate to the opposite side of the proton-transfer pathway by constructing the double mutant EA(I-286)/IE(I-112). This places the side chain in about the same position in space as in the original enzyme, but does not allow for the same type of movement. The results show that the introduction of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In addition, the double mutant enzyme pumps approximately 0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation of the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific location in space, presumably to stabilize an intact water chain. However, this residue does not necessarily have to be at a strictly conserved location in the amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumped protons from one to the other side of the membrane.
Aagaard
A
A
Department of Biochemistry, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
Gilderson
G
G
Mills
D A
DA
Ferguson-Miller
S
S
Brzezinski
P
P
D002384:Catalysis; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017494:Proton Pumps / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology
D017494:Proton Pumps; D011994:Recombinant Proteins; D018698:Glutamic Acid; D003576:Electron Transport Complex IV