Cyanide and Carbon Monoxide Binding to the Reduced Form of Cytochrome bo from Escherichia coli

Abstract

Cyanide binds to fully reduced cytochrome bo and induces a blue shift of the Soret absorption band of the high-spin heme o and a change in the visible region spectrum consistent with the expected conversion to a low-spin state. The dissociation constant, determined by titration of the extent of the binding spectrum, is 7.0 +/- 0.6 mM at pH 7.0. In contrast, cyanide does not bind significantly in this concentration range to the reduced form of cytochrome bd. The reduced cyanide compound of cytochrome bo can be laser photolyzed. Typically, less than 20% photolysis was attained with conditions that give essentially full photolysis of the carbon monoxide compound. The subsequent monophasic kinetics of recombination of cyanide at varying cyanide concentrations were used to determine kon, koff, and dissociation constant values at pH 7.0 of 572 +/- 43 M-1 s-1, 4.2 +/- 0.7 s-1, and 7.3 +/- 1.3 mM, respectively. The dissociation constant changes very little in the pH range 6-8, indicating that a proton is bound together with the cyanide anion, as predicted by our recent proposal of a requirement for electroneutrality in the binuclear center [Mitchell, R.,& Rich, P. R. (1994) Biochim. Biophys. Acta 1186, 19-26]. Competition studies confirm that cyanide and carbon monoxide cannot bind simultaneously, so that their binding sites must overlap. A small fraction of the reduced unliganded enzyme appears to have a distinct photolysis spectrum in the absence of added ligands, and this is transformed into a typical ferrous cyanide compound only at very high cyanide concentrations. Cyanide binding and photolysis were also examined in a number of mutant forms of cytochrome bo, and in a wild-type form which was partially depleted in CuB. Dramatic changes in rate constants and binding constants were found in several cases. Data from several mutants were compared with analogous data on the binding and photolysis of carbon monoxide, and the effects of mutation were quite different with the two ligands. A model is developed to explain the observed effects of point mutations on the recombination kinetics of both carbon monoxide and cyanide. Overall, the results indicate that the CuB site is required for binding of cyanide, but not carbon monoxide, to the reduced enzyme, possibly by providing the site for binding of the associated proton.

Comparison of the ligand-binding properties of native and copper-less cytochromes bo from Escherichia coli

“…Laser-flash photolysis was performed with a frequency-doubled Nd-YAG laser (Spectron Ltd., Rugby, Warwickshire, U.K.) as described before [15].…”

“…The CO-binding spectra of fully reduced membranes, which are identical for kCu and jCu membranes (isolated from E. coli cells grown respectively without a copper supplement and with a copper chelator present, and with a copper supplement) [15], were used to determine the relative cytochrome bo content ;…”

“…was used, for which the absorption coefficients were taken to be 7 and 196 mM −" :cm −" respectively [15]. Routinely, however, after ligand binding the sample was reduced (for 1 min with 10 mM dithionite), and a dithionite-reduced minus air-oxidized (redox) spectrum generated.…”

“…One approach to the latter is to study site-directed mutants chosen with the aid of the accumulated structural knowledge of these enzymes that now includes high-resolution X-ray crystallographic information [3][4][5]. In this paper, however, we take advantage of the finding that wild-type cytochrome bo can assemble without Cu B when E. coli is grown under copper-limited conditions [12][13][14][15]. We…”

EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB

“…These are almost identical for Cu(+) and Cu(-) membranes [12]. The mean of AA536_556mn an d AA57o-590nm was used in this determination, for which the extinction coefficient was taken to be approximately 7 mM -1 cm -1 [12].…”