title: Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway.
abstract: IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron, and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, although the products differ from those generated by canonical heme oxygenases. A synthesis of time-resolved techniques, including in proteo mass spectrometry and conventional and stopped-flow UV/visible spectroscopy, was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso-hydroxyheme (forming with k = 0.6 min(-1), pH 7.4, 10 mm ascorbate, 10 μm IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases. Unlike heme oxygenases, this intermediate does not form with added H2O2 nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min(-1)) were a set of formyloxobilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to β-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG's dual role as both an oxygenase and a sensor of heme availability in S. aureus.
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Bennett R
authors.initials: BR
authors.lastname: Streit
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Ravi
authors.initials: R
authors.lastname: Kant
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Monika
authors.initials: M
authors.lastname: Tokmina-Lukaszewska
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Arianna I
authors.initials: AI
authors.lastname: Celis
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Melodie M
authors.initials: MM
authors.lastname: Machovina
authors.affiliation: the Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine and the Tennessee Valley Healthcare Systems, United States Department of Veterans Affairs, Nashville, Tennessee 37232.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Skaar
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: From the Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59715-3400, jdubois@chemistry.montana.edu.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: DuBois
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism; D002853:Chromatography, Liquid; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007554:Isotopes; D007700:Kinetics; D013058:Mass Spectrometry; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000201:enzymology; D013997:Time Factors
chemical_list: D007554:Isotopes; D006418:Heme; D006861:Hydrogen Peroxide; D010105:Oxygenases; C482079:IsdG protein, Staphylococcus aureus; D006419:Heme Oxygenase (Decyclizing); D001205:Ascorbic Acid; D010100:Oxygen
keywords: Staphylococcus aureus (S. aureus); carbon monoxide; heme; heme oxygenase; iron; tetrapyrrole