Fourier transform infrared evidence for connectivity between Cu-B and glutamic acid 286 in cytochrome bo(3) from Escherichia coli

Photodissociation of fully reduced, carbonmonoxy cytochrome bo(3) causes ultrafast transfer of carbon monoxide (C equivalent to O) from heme iron to Cu-B in the binuclear site. At low temperatures, the C equivalent to O remains bound to Cu-B for extended times. Here, we show that the binding of C equivalent to O to CUB perturbs the IR stretch of an un-ionized carboxylic acid residue, which is identified as Glu286 by mutation to Asp or to Cys. Before photodissociation, the carbonyl (C=O)-stretching frequency of this carboxylic acid residue is 1726 cm(-1) for Glu286 and 1759 cm(-1) for Glu286Asp. These frequencies are definitive evidence for un-ionized R-COOH and suggest that the carboxylic acids are hydrogen-bonded, though more extensively in Glu286. In Glu286Cys, this IR feature is lost altogether, We ascribe the frequency shifts in the C=O IR absorptions to the effects of binding photodissociated C equivalent to O to Cu-B, which are relayed to the 286 locus, Conversely, the 2065 cm(-1) C equivalent to O stretch of Cu-B-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of Cu-B, or to displacement of a Cu-B histidine ligand by C equivalent to O. C equivalent to O binding to Cu-B also induces a downshift of an IR band which can be attributed to an aromatic C-H stretch, possibly of histidine imidazole, at about 3140 cm(-1) The results suggest an easily polarizable, through-bond connectivity between one of the histidine Cu-B ligands and the carboxylic group of Glu286. A chain of bound water molecules may provide such a connection, which is of interest in the context of the proton pump mechanism of the heme-copper oxidases.