title: Site-directed mutation of the highly conserved region near the Q-loop of the cytochrome bd quinol oxidase from Escherichia coli specifically perturbs heme b595.
abstract: Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV--vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Hellwig
authors.forename: J P
authors.initials: JP
authors.lastname: Osborne
authors.forename: H W
authors.initials: HW
authors.lastname: Huang
authors.forename: P
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.forename: A A
authors.initials: AA
authors.lastname: Konstantinov
authors.forename: R B
authors.initials: RB
authors.lastname: Gennis
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D002248:Carbon Monoxide / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics; D003486:Cyanides / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D018698:Glutamic Acid / Q000235:genetics; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011808:Quinone Reductases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman
chemical_list: D003486:Cyanides; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; C435883:heme b(595); D018698:Glutamic Acid; D006418:Heme; D002248:Carbon Monoxide; D010088:Oxidoreductases; D010089:Oxidoreductases, N-Demethylating; C022726:tetramethylphenylenediamine oxidase; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase; C040077:cytochrome bd terminal oxidase complex, E coli; D000409:Alanine