title: Spectroscopic studies on the active site of hydroperoxide lyase; the influence of detergents on its conformation.
abstract: Expression of high quantities of alfalfa hydroperoxide lyase in Escherichia coli made it possible to study its active site and structure in more detail. Circular dichroism (CD) spectra showed that hydroperoxide lyase consists for about 75% of alpha-helices. Electron paramagnetic resonance (EPR) spectra confirmed its classification as a cytochrome P450 enzyme. The positive influence of detergents on the enzyme activity is paralleled by a spin state transition of the heme Fe(III) from low to high spin. EPR and CD spectra showed that detergents induce a subtle conformational change, which might result in improved substrate binding. Because hydroperoxide lyase is thought to be a membrane bound protein and detergents mimic a membrane environment, the more active, high spin form likely represents the in vivo conformation. Furthermore, the spin state appeared to be temperature-dependent, with the low spin state favored at low temperature. Point mutants of the highly conserved cysteine in domain D indicated that this residue might be involved in heme binding.
authors.affiliation: Bijvoet Center for Biomolecular Research, Department of Bio-organic Chemistry, Utrecht University, Padualaan 8, NL-3584 CH, Utrecht, The Netherlands.
authors.forename: M A
authors.initials: MA
authors.lastname: Noordermeer
authors.forename: G A
authors.initials: GA
authors.lastname: Veldink
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D000455:Medicago sativa / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation / Q000187:drug effects
chemical_list: D003902:Detergents; D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase