1f8w

CRYSTAL STRUCTURE OF NADH PEROXIDASE MUTANT: R303M

spinqualitylabelsall models 1f8w, resolution 2.45Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 1f8w is a 1 chain structure with sequence from "enterococcus_proteiformis"_thiercelin_and_jouhaud_1903 "enterococcus proteiformis" thiercelin and jouhaud 1903. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: FAD NonStd Res: CSX Related: 1joa, 1nhp, 1nhq, 1nhr, 1nhs, 1npx Activity: NADH peroxidase, with EC number 1.11.1.1 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function [NAPE_ENTFA] Peroxidase whose active site is a redox-active cysteine-sulfenic acid. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also NADH peroxidase

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