Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation

• Published in: Journal of the American Chemical Society Vol. 134; no. 27; pp. 11225 - 11234
• Main Authors: Wang, Weixue, Wang, Ke, Span, Ingrid, Jauch, Johann, Bacher, Adelbert, Groll, Michael, Oldfield, Eric
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 11.07.2012; Amer Chemical Soc
• Subjects: Betula; biosynthesis; catalytic activity; Chemistry; Chemistry, Multidisciplinary; Crystallography, X-Ray; deuterium; drugs; electron paramagnetic resonance spectroscopy; Electron Spin Resonance Spectroscopy; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; free radicals; Free Radicals - metabolism; isoprenoids; isotope labeling; mechanism of action; Models, Molecular; Mutation; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; phosphates; Physical Sciences; Science & Technology; stable isotopes; thioredoxins; X-ray diffraction; DEOXYXYLULOSE PHOSPHATE-PATHWAY; MACROMOLECULAR STRUCTURES; SUBSTRATE-BINDING; DOUBLE-RESONANCE CHARACTERIZATION; ISOPRENOID BIOSYNTHESIS; (E)-1-HYDROXY-2-METHYLBUT-2-ENYL 4-DIPHOSPHATE; (E)-4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE; NON-MEVALONATE PATHWAY; THIOREDOXIN REDUCTASE; INHIBITOR BINDING
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja303445z

The [4Fe–4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and 2H, 17O, and 57Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe–4S]2+ cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron–sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate’s 3-CH2OH group has rotated away from the reduced iron–sulfur cluster, and the next, η3-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.