Backbone NMR Assignments and H/D Exchange Studies on the Ferric Azide- and Cyanide-Inhibited Forms of Pseudomonas aeruginosa Heme Oxygenase

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• 作者：Juan Carlos Rodrí ; guez ; Angela Wilks ; and Mario Rivera
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：April 11, 2006
• 年：2006
• 卷：45
• 期：14
• 页码：4578 - 4592
• 全文大小：708K
• 年卷期：v.45,no.14(April 11, 2006)
• ISSN：1520-4995

文摘

The 198 amino acid long heme oxygenase from Pseudomonas aeruginosa (pa-HO) was studiedby multinuclear and multidimensional NMR spectroscopy in its paramagnetic cyanide-inhibited (pa-HO-CN) and azide-inhibited (pa-HO-N₃) forms. Nearly complete backbone assignments (>93%) of all non-proline residues have been obtained, with the majority of the nonassigned residues corresponding to thefirst 10 amino terminal residues. Resonances strongly affected by heme iron paramagnetism were assignedwith the aid of selective amino acid labeling and experiments tailored to detect fast relaxing signals,whereas the rest of the polypeptide was assigned using conventional three-dimensional NMR experiments.Amide chemical shift assignments were used to monitor the rate of exchange of backbone protons inhydrogen-deuterium exchange experiments. The polypeptide in the pa-HO-N₃ complex was found tobe significantly less prone to exchange than the polypeptide in pa-HO-CN, which we interpret to indicatethat pa-HO-N₃ is conformationally less flexible than pa-HO-CN. The differences in protection factorsextend to regions of the protein remote from the heme iron and distal ligand. Mapping the differences inprotection factors into the X-ray crystal structure of pa-HO [Friedman, J., Lad, L., Li, H., Wilks, A.Poulos, T. L. (2004) Biochemistry 43, 5239-5345] suggests that the distinct chemical properties impartedby the coordination of azide or cyanide to the heme iron [Zeng, Y. Caignan, G. A., Bunce, R. A., Rodríguez,J. C., Wilks, A., Rivera, M. (2005) J. Am. Chem. Soc. 127, 9794-9807] are transmitted to the polypeptideby a network of structural water molecules extending from the active site to the surface of the enzyme.Finally, while the ¹H amide resonance of Gly125 was too broad to detect, the corresponding ¹⁵N resonanceexhibits a large downfield shift, large line width, steep temperature dependence, and a larger than usualupfield deuterium isotope effect. These properties indicate unpaired spin delocalization from the hemeiron into the Gly ¹⁵N atom via formation of a hydrogen bond between the coordinated azide nitrogen andthe Gly125 N-H.