| Autor: |
Puustinen A; Bioscience and Biotechnology Group (CST-4), Chemical Sciences and Technology Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA., Bailey JA, Dyer RB, Mecklenburg SL, Wikström M, Woodruff WH |
| Abstrakt: |
Photodissociation of fully reduced, carbonmonoxy cytochrome bo3 causes ultrafast transfer of carbon monoxide (C triple bond O) from heme iron to CuB in the binuclear site. At low temperatures, the C triple bond O remains bound to CuB for extended times. Here, we show that the binding of C triple bond 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 triple bond O to CuB, which are relay ed to the 286 locus. Conversely, the 2065 cm-1 C triple bond O stretch of CuB-CO is markedly affected by both mutations. These effects are ascribed to changes in the Lewis acidity of CuB, or to displacement of a CuB histidine ligand by C triple bond O. C triple bond O binding to CuB 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 CuB 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. |
