Structural Studies of the Molybdenum Center of Mitochondrial Amidoxime Reducing Component (mARC) by Pulsed EPR Spectroscopy and 17O-Labeling

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• 作者：Asha Rajapakshe ; Andrei V. Astashkin ; Eric L. Klein ; Debora Reichmann ; Ralf R. Mendel ; Florian Bittner ; John H. Enemark
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：October 18, 2011
• 年：2011
• 卷：50
• 期：41
• 页码：8813-8822
• 全文大小：404K
• 年卷期：v.50,no.41(October 18, 2011)
• ISSN：1520-4995

文摘

Mitochondrial amidoxime reducing components (mARC-1 and mARC-2) represent a novel group of Mo-containing enzymes in eukaryotes. These proteins form the catalytic part of a three-component enzyme complex known to be responsible for the reductive activation of several N-hydroxylated prodrugs. No X-ray crystal structures are available for these enzymes as yet. A previous biochemical investigation [Wahl, B., et al. (2010) J. Biol. Chem., 285, 37847鈥?7859 ] has revealed that two of the Mo coordination positions are occupied by sulfur atoms from a pyranopterindithiolate (molybdopterin, MPT) cofactor. In this work, we have used continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy and density functional theoretical (DFT) calculations to determine the nature of remaining ligands in the Mo(V) state of the active site of mARC-2. Experiments with samples in D₂O have identified the exchangeable equatorial ligand as a hydroxyl group. Experiments on samples in H₂¹⁷O-enriched buffer have shown the presence of a slowly exchangeable axial oxo ligand. Comparison of the experimental ¹H and ¹⁷O hyperfine interactions with those calculated using DFT has shown that the remaining nonexchangeable equatorial ligand is, most likely, protein-derived and that the possibility of an equatorial oxo ligand can be excluded.