The binding of cyanide and carbon monoxide to bacterial cytochrome bo

Abstract

Introduction Cytochrome (cyt) bo from Exherichia coli is a member of a superfamily of homologous protonmotive oxidases which includes mammalian cyt c oxidase [ l ] . It has become evident that the copperhaem binuclear centre is very similar in all of these oxidases even though the haem type and reducing substrate vary. This binuclear centre is the site of oxygen binding and reduction to water, and the notion that local ligand movements in the binuclear centre drive proton translocation between proton channels in the protein structure [21 has become widely adopted as the most likely working model for the primary chemistry that drives vectorial proton translocation (for detailed variants on this theme, see [ 3-71). We have embarked on a detailed study of ligand binding dynamics in order to gain further insight into such possible protonmotive chemistry. Site-directed mutagenesis in bacterial systems o f conserved possible metal ligands allowed fairly definitive assignment of four histidine ligands to the three metal centres (two haem groups and one copper, Cu,,) in the critical subunit I of these oxidases [ X,9 1. Further biophysical analyses led to reassignment of the two remaining conserved histidines of subunit 1, together with the identification of other amino acids which might influence the metal centre chemistry. A summary of the view reached by our own laboratory of the possible ligands around Cu,,. based on data in [X,O] and on our measurements of mutation effects on recombination o f carbon monoxide after laser photolysis [lo], is shown in Figure 1. Complementary types of spectroscopic data from other laboratories have produced a remarkably consistent model [ 11-13]. We found that mutation of two histidine ligands (H333I,, H3341,) led to an acceleration of the rate of recombination of carbon monoxide with the binuclear centre [lo], a result also observed if the wild-type enzyme is grown in copper-limited conditions so that Cull is absent [14,15]. We have deduced that an acceleration of recombination rate of fivefold or more can be taken as a strong indication that Cu,, is absent. Indeed, we have often observed heterogeneity of CO recombination in