Cytochrome bo from Escherichia coli: identification of haem ligands and reaction of the reduced enzyme with carbon monoxide

Abstract

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].

Molecular and Spectroscopic Analysis of the Cytochromecbb3 Oxidase from Pseudomonas stutzeri

“…However, a signal at g ϭ 6.02 must arise from a small amount of uncoupled high-spin ferric heme b 3 . This can be explained in terms of a small proportion of the preparation being damaged or lacking Cu B , something that we have previously observed for cytochrome bo 3 from E. coli (41). This view is consistent with our observation that the X-band EPR spectrum of one preparation of cytochrome cbb 3 contained a much larger high-spin ferric heme signal (data not shown).…”

Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath)

“…The nitric oxide derivatives of ascorbate-PMS-reduced samples were prepared by using the in situ reduction of the nitrite ion as the source of the nitric oxide (11). The protein-NO complex was formed anaerobically with reduced samples by adding solid H 14 NO 2 or H 15 NO 2 (ICL, Andover, Mass.)…”

The Reaction of Hydrogen Peroxide with Pulsed Cytochrome bo from Escherichia coli

“…octyl-P-D-glucopyranoside for 30 min at 4°C followed by centrifugation (150000 g,, for 45 min at 4°C) to remove insoluble material. Further purification of the enzyme on DEAESepharose CL-6B and o-aminohexyl agarose columns was essentially as described by Cheesman et al (1993). Note, however, that the detergent exchange step at the end, to change the detergent from Triton X-100 to OCtyl-P-D-glUC0-pyranoside, was not carried out.…”