Title

Backbone NMR assignments and H/D exchange studies on the ferric azide- and cyanide-inhibited forms of Pseudomonas aeruginosa heme oxygenase

Document Type

Article

Publication Date

4-11-2006

Abstract

The 198 amino acid long heme oxygenase from Pseudomonas aeruginosa (pa-HO) was studied by multinuclear and multidimensional NMR spectroscopy in its paramagnetic cyanide-inhibited (pa-HO-CN) and azide-inhibited (pa-HO-N 3) forms. Nearly complete backbone assignments (>93%) of all non-proline residues have been obtained, with the majority of the nonassigned residues corresponding to the first 10 amino terminal residues. Resonances strongly affected by heme iron paramagnetism were assigned with 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 in hydrogen-deuterium exchange experiments. The polypeptide in the pa-HO-N3 complex was found to be significantly less prone to exchange than the polypeptide in pa-HO-CN, which we interpret to indicate that pa-HO-N3 is conformationally less flexible than pa-HO-CN. The differences in protection factors extend to regions of the protein remote from the heme iron and distal ligand. Mapping the differences in protection 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 imparted by 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 polypeptide by a network of structural water molecules extending from the active site to the surface of the enzyme. Finally, while the 1H amide resonance of Gly 125 was too broad to detect, the corresponding 15N resonance exhibits a large downfield shift, large line width, steep temperature dependence, and a larger than usual upfield deuterium isotope effect. These properties indicate unpaired spin delocalization from the heme iron into the Gly 15N atom via formation of a hydrogen bond between the coordinated azide nitrogen and the Gly 125 N-H. © 2006 American Chemical Society.

Publication Source (Journal or Book title)

Biochemistry

First Page

4578

Last Page

4592

This document is currently not available here.

Share

COinS