Cytochrome bo from Escherichia coli: identification of haem ligands and reaction of the reduced enzyme with carbon monoxide.
Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [Au and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells. Optical e.p.r. and low-temperature m.c.d. (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E. coli. Low-spin ferric haem b, the origin of a rhombic e.p.r. spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r. m.c.d. spectrum at 1622 nm. It is therefore concluded that haem b is co-ordinated by two histidine residues. The low-temperature m.c.d. spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o. The bands arising from haem o show a direct correspondence with those in the m.c.d. spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine. This assignment was confirmed by measuring the e.p.r. spectrum of the nitric oxide derivative of fully reduced cytochrome bo. This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine. The hyperfine splittings observed were 1.95 +/- 0.05 mT for the 14N of the NO ligand and 0.75 +/- 0.05 mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6. These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-CuB, suggesting that the binuclear site may display heterogeneity. Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1, and the dissociation constant for this reaction was determined to be 1.7 x 10(-6)M. The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 [Gibson and Greenwood (1963) Biochem. J. 86, 541-554].
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, U.K.
M R
MR
Cheesman
N J
NJ
Watmough
C A
CA
Pires
R
R
Turner
T
T
Brittain
R B
RB
Gennis
C
C
Greenwood
A J
AJ
Thomson
D002248:Carbon Monoxide / Q000378:metabolism*; D003300:Copper / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D006418:Heme / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008667:Metalloproteins / Q000737:chemistry*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D008024:Ligands; D008667:Metalloproteins; D009569:Nitric Oxide; D006418:Heme; D003300:Copper; D002248:Carbon Monoxide; C017168:cytochrome bo, E coli; D007501:Iron