Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway.

Streit BR, Kant R, Tokmina-Lukaszewska M, Celis AI, Machovina MM, Skaar EP, Bothner B, DuBois JL
J Biol Chem. 2016 291 (2): 862-71

PMID: 26534961 · PMCID: PMC4705404 · DOI:10.1074/jbc.M115.666560

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.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (15)

Ascorbic Acid Chromatography, Liquid Heme Heme Oxygenase (Decyclizing) Humans Hydrogen Peroxide Isotopes Kinetics Mass Spectrometry Oxygen Oxygenases Signal Transduction Spectrophotometry, Ultraviolet Staphylococcus aureus Time Factors

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