TY - JOUR
T1 - Cytochrome bo from Escherichia coli
T2 - Identification of haem ligands and reaction of the reduced enzyme with carbon monoxide
AU - Cheesman, M. R.
AU - Watmough, N. J.
AU - Pires, C. A.
AU - Turner, R.
AU - Brittain, T.
AU - Gennis, R. B.
AU - Greenwood, C.
AU - Thomson, A. J.
PY - 1993
Y1 - 1993
N2 - Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [An and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl β-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±0.05mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15K, 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-Cu(B), 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].
AB - Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [An and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl β-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±0.05mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15K, 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-Cu(B), 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].
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U2 - 10.1042/bj2890709
DO - 10.1042/bj2890709
M3 - Article
C2 - 8382047
AN - SCOPUS:0027533344
SN - 0264-6021
VL - 289
SP - 709
EP - 718
JO - Biochemical Journal
JF - Biochemical Journal
IS - 3
ER -