Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.
Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.
Mitchell
D M
DM
School of Chemical Sciences, University of illinois, Urbana 61801, USA.
Fetter
J R
JR
Mills
D A
DA
Adelroth
P
P
Pressler
M A
MA
Kim
Y
Y
Aasa
R
R
Brzezinski
P
P
Malmström
B G
BG
Alben
J O
JO
Båbcock
G T
GT
Ferguson-Miller
S
S
Gennis
R B
RB
D002248:Carbon Monoxide / Q000378:metabolism; D017124:Conserved Sequence; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV