title: Fourier transform infrared evidence for connectivity between CuB and glutamic acid 286 in cytochrome bo3 from Escherichia coli.
abstract: 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.
authors.affiliation: Bioscience and Biotechnology Group (CST-4), Chemical Sciences and Technology Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
authors.forename: A
authors.initials: A
authors.lastname: Puustinen
authors.forename: J A
authors.initials: JA
authors.lastname: Bailey
authors.forename: R B
authors.initials: RB
authors.lastname: Dyer
authors.forename: S L
authors.initials: SL
authors.lastname: Mecklenburg
authors.forename: M
authors.initials: M
authors.lastname: Wikström
authors.forename: W H
authors.initials: WH
authors.lastname: Woodruff
mesh_terms: D003300:Copper / Q000737:chemistry*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006639:Histidine; D006860:Hydrogen Bonding; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D008024:Ligands; C083850:cytochrome bo3, E coli; D018698:Glutamic Acid; D006639:Histidine; D003300:Copper