Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer.
The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.
Wang
Kefei
K
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Geren
Lois
L
Zhen
Yuejun
Y
Ma
Ling
L
Ferguson-Miller
Shelagh
S
Durham
Bill
B
Millett
Francis
F
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003300:Copper / Q000378:metabolism*; D004579:Electron Transport / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics; D008024:Ligands; D008715:Methionine / Q000235:genetics; D009942:Organometallic Compounds / Q000737:chemistry; D010782:Photolysis; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics*; D012428:Ruthenium / Q000737:chemistry; D014461:Ultracentrifugation / Q000379:methods
D008024:Ligands; D009942:Organometallic Compounds; D006639:Histidine; D001216:Asparagine; D003300:Copper; D012428:Ruthenium; D008715:Methionine; D003576:Electron Transport Complex IV; D007930:Leucine