The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN

Layer, Gunhild and Pierik, Antonio J. and Trost, Matthias and Rigby, Stephen E. J. and Leech, Helen K. and Grage, Katrin and Breckau, Daniela and Astner, Isabel and Jansch, Lothar and Heathcote, Peter and Warren, Martin J. and Heinz, Dirk W. and Jahn, Dieter (2006) The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN. Journal of Biological Chemistry, 281 (23). pp. 15727-34. ISSN 0021-9258. (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1074/jbc.M512628200

Abstract

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.

Item Type: Article
Additional information: 0021-9258 (Print) Journal Article Research Support, Non-U.S. Gov't
Uncontrolled keywords: Bacillus megaterium/enzymology Bacterial Proteins/*metabolism Chromatography, High Pressure Liquid Coproporphyrinogen Oxidase/*metabolism Electron Spin Resonance Spectroscopy Escherichia coli/*enzymology Oxygen/*metabolism Substrate Specificity
Subjects: Q Science
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: Martin Warren
Date Deposited: 29 Jun 2011 16:51
Last Modified: 17 Jul 2014 09:23
Resource URI: http://kar.kent.ac.uk/id/eprint/11036 (The current URI for this page, for reference purposes)
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