1f8w

From Proteopedia

Revision as of 12:35, 20 November 2007

CRYSTAL STRUCTURE OF NADH PEROXIDASE MUTANT: R303M

Overview

The crystal structure of the flavoprotein NADH peroxidase shows that the, Arg303 side chain forms a hydrogen bond with the active-site His10, imidazole and is therefore likely to influence the catalytic mechanism., Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)], yields a two-electron reduced intermediate (EH(2)) with enhanced flavin, fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a), for the nascent Cys42-SH is increased by over 3.5 units in comparison with, the wild-type EH(2) pK(a) of </=4.5. NADH titration of the mutant, peroxidase yields the same EH(2) intermediate, but in contrast to the, behavior of wild-type enzyme, this species can be reduced directly to an, EH(4).NAD(+) complex. Kinetic analyses demonstrate that the R303M mutant, is severely compromised, although active, with k(cat) = 3 s(-)(1) at pH, 7.0, 5 degrees C; enzyme-monitored turnover results indicate that the, steady-state consists predominantly of an E-FADH(2).NAD(+) species. When, the oxidized mutant is reacted anaerobically with 0.9 equiv of NADH/FAD, a, clearly biphasic pattern is observed at 450 nm; relatively rapid flavin, reduction is followed by reoxidation at 2.6-2.7 s(-)(1) ( approximately, k(cat)). Thus replacement of Arg303 with Met leads to an altered, peroxidase form in which the rate-limiting step in turnover is the, intramolecular transfer of electrons from FADH(2) --> Cys42-SOH. The, crystal structure of the R303M peroxidase has been refined at 2.45 A, resolution. In addition to eliminating the Arg303 interactions with His10, and Glu14, the mutant exhibits a significant change in the conformation of, the Cys42-SOH side chain relative to FAD and His10 in particular. These, and other results provide a detailed understanding of Arg303 and its role, in the structure and mechanism of this unique flavoprotein peroxidase.

About this Structure

1F8W is a Single protein structure of sequence from Enterococcus faecalis with FAD as ligand. Active as NADH peroxidase, with EC number 1.11.1.1 Full crystallographic information is available from OCA.

Reference

Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure., Crane EJ 3rd, Yeh JI, Luba J, Claiborne A, Biochemistry. 2000 Aug 29;39(34):10353-64. PMID:10956025

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