Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli.
The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature. The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively. Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis. A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals. Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I. In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem. This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1. The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB. It appeared that rapid haem-haem electron transfer could still occur. The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase.
Brown
S
S
Glynn Research Institute, Bodmin, UK.
Rumbley
J N
JN
Moody
A J
AJ
Thomas
J W
JW
Gennis
R B
RB
Rich
P R
PR
D000595:Amino Acid Sequence; D002248:Carbon Monoxide / Q000737:chemistry*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010782:Photolysis
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D002248:Carbon Monoxide; C017168:cytochrome bo, E coli