Ligand-binding properties and heterogeneity of cytochrome bo from Escherichia coli

• Published in: Biochimica et biophysica acta Vol. 1141; no. 2-3; p. 321
• Main Authors: Moody, A J, Rumbley, J N, Gennis, R B, Ingledew, W J, Rich, P R
• Format: Journal Article
• Language: English
• Published: Netherlands 01-03-1993
• Subjects: Cyanides - chemistry; Electron Transport Complex IV - chemistry; Electron Transport Complex IV - isolation & purification; Electron Transport Complex IV - metabolism; Escherichia coli - enzymology; Formates - chemistry; Kinetics; Ligands; Protein Binding; Spectrophotometry, Ultraviolet
• ISSN: 0006-3002
• DOI: 10.1016/0005-2728(93)90060-S

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 degrees 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 degrees 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.