Research Article
A temperature-dependent conformational change of NADH oxidase from Thermus thermophilus HB8 †
Eric D. Merkley,
Valerie Daggett,
William W. Parson,
Eric D. Merkley
Department of Biochemistry, University of Washington, Seattle, Washington
Search for more papers by this authorValerie Daggett
Department of Biochemistry, University of Washington, Seattle, Washington
Department of Bioengineering, University of Washington, Seattle, Washington
Search for more papers by this authorCorresponding Author
William W. Parson
Department of Biochemistry, University of Washington, Seattle, Washington
Department of Biochemistry, University of Washington, Box 357351, Seattle, WA 98195-7350===Search for more papers by this authorEric D. Merkley,
Valerie Daggett,
William W. Parson,
Eric D. Merkley
Department of Biochemistry, University of Washington, Seattle, Washington
Search for more papers by this authorValerie Daggett
Department of Biochemistry, University of Washington, Seattle, Washington
Department of Bioengineering, University of Washington, Seattle, Washington
Search for more papers by this authorCorresponding Author
William W. Parson
Department of Biochemistry, University of Washington, Seattle, Washington
Department of Biochemistry, University of Washington, Box 357351, Seattle, WA 98195-7350===Search for more papers by this author†
Eric D. Merkley's current address is Pacific Northwest Research Laboratory, PO Box 999 MSIN: K8-98 Richland, WA 99352
Abstract
Using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far-UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature-dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.
Supporting Information
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