title: Introduction of water into the heme distal side by Leu65 mutations of an oxygen sensor, YddV, generates verdoheme and carbon monoxide, exerting the heme oxygenase reaction.
abstract: The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.
authors.forename: Martin
authors.initials: M
authors.lastname: Stranava
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic. Electronic address: marketa.martinkova@natur.cuni.cz.
authors.forename: Markéta
authors.initials: M
authors.lastname: Martínková
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic; Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, Prague 4, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kitanishi
authors.affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, 1(st) Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Muchová
authors.affiliation: Institute of Medical Biochemistry and Laboratory Diagnostics, 1(st) Faculty of Medicine, Charles University in Prague, Czech Republic.
authors.forename: Libor
authors.initials: L
authors.lastname: Vítek
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.
authors.forename: Václav
authors.initials: V
authors.lastname: Martínek
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova (Albertov) 2030/8, Prague 2, 128 43, Czech Republic.
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D002248:Carbon Monoxide / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D007930:Leucine / Q000235:genetics*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D014867:Water; D006418:Heme; D002248:Carbon Monoxide; D006419:Heme Oxygenase (Decyclizing); D019761:Phosphorus-Oxygen Lyases; C553251:yddV protein, E coli; D007930:Leucine; D010100:Oxygen
keywords: Diguanylate cyclase; Globin-coupled oxygen sensor; Heme oxygenase; Heme-based oxygen sensor; NADPH:cytochrome P450 reductase; Verdoheme