FOURIER-TRANSFORM INFRARED EVIDENCE FOR CONNECTIVITY BETWEEN CU-B ANDGLUTAMIC-ACID-286 IN CYTOCHROME BO(3) FROM ESCHERICHIA-COLI

Photodissociation of fully reduced, carbonmonoxy cytochrome bo(3) caus es ultrafast transfer of carbon monoxide (C equivalent to O) from heme iron to Cu-B in the binuclear site. At low temperatures, the C equiva lent 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 defini tive evidence for un-ionized R-COOH and suggest that the carboxylic ac ids are hydrogen-bonded, though more extensively in Glu286. In Glu286C ys, this IR feature is lost altogether, We ascribe the frequency shift s 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, Conver sely, 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 liga nd by C equivalent to O. C equivalent to O binding to Cu-B also induce s 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 re sults 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 hem e-copper oxidases.