Cyanide and formate induce spectral changes in E. coli cytochrome bo which are similar to those induced in bovine heart cytochrome-c oxidase (cytochrome aa3). Cyanide induces a red shift of 6 nm in the Soret band, whereas formate induces a blue shift of 2 nm. Cytochrome bo as purified shows multiphasic cyanide-binding kinetics. At least three phases can be seen with rate constants of 16, 1 and 0.1 M-1 s-1, respectively, at pH 7 and 20°C. The enzyme after redox cycling ('pulsing') or in situ in E. coli membranes shows essentially monophasic binding with a rate constant of 15 M-1 s-1. Further evidence of heterogeneity in the enzyme as prepared comes from formate binding, which also shows at least three phases (rate constants of 1.4, 0.2 and 0.01 M-1 s-1, respectively, at pH 5 and 20°C). The fast phase of cyanide binding is eliminated in less than 2 min by incubation with 40 mM formate, but the intermediate phase is unaffected by incubation for 3.5 h with 40 mM formate. Thus, the subpopulation that causes the fast phase of cyanide binding also causes the fast phase of formate binding. Formate-ligated cytochrome bo has similar cyanide-binding kinetics to the subpopulation that causes the slow phase of cyanide binding in cytochrome bo as prepared. It appears, from all this, that the subpopulations responsible for the fast and slow phase of cyanide binding are analogous to the 'fast' and 'slow' forms, respectively, of cytochrome aa3. However, there is no spontaneous conversion of the 'fast' form of cytochrome bo to the 'slow' form at low pH (5.1). The rate of cyanide binding to cytochrome bo during turnover is much greater (k > 103 M-1 s-1) than the rate of binding to the fully-oxidised enzyme (k < 20 M-1 s-1). Hence, like cytochrome aa3, cytochrome bo shows the 'closed'/'open' phenomenon.
Bibliographical noteFunding Information:
This work was supported by the SERC (grant GR/F/17605 awarded to P.R.R. and grant GR/F/ 55348 awarded to W.J.I. and Prof. D.H. Boxer). We are indebted to other benefactors, named in the brochure of the U.K. Registered Charity called the Glynn Research Foundation Ltd, for general financial support of the research facilities at the Glynn Research Institute. We are grateful for the technical assistance of Sally Madgwick, Robert Harper and Alan Jeal at Glynn and Alex Houston at St. Andrews.
- (E. coli)
- Cytochrome bo