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J. Biol. Chem., Vol. 282, Issue 24, 17767-17776, June 15, 2007

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Exploring Molecular Oxygen Pathways in Hansenula polymorpha Copper-containing Amine Oxidase^*

Bryan J. Johnson, Jordi Cohen, Richard W. Welford^¶, Arwen R. Pearson, Klaus Schulten, Judith P. Klinman^¶, and Carrie M. Wilmot¹

From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, Beckman Institute, University of Illinois, Urbana-Champaign, Illinois 61801, and ^¶Departments of Chemistry and Molecular and Cellular Biology, University of California, Berkeley, California 94720

The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O₂ free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O₂ migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k_cat/K_m (O₂), supporting multiple dynamic pathways for molecular oxygen to reach the active site.

Received for publication, February 14, 2007 , and in revised form, March 26, 2007.

The atomic coordinates and structure factors (codes 2oqe and 2oov) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Training Grant GM-008700 (to B. J. J.), National Institutes of Health Grants GM-66569 (to C. M. W.), GM-25765 (to J. P. K.), and P41-RR05969 (to K. S.), Minnesota Medical Foundation Grant 3714-9221-06 (to C. M. W.), and National Science Foundation supercomputer time grant NRAC MCA93S028. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3, Figs. S1–S3, and data files.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455. Tel.: 612-624-2406; Fax: 612-624-5121; E-mail: wilmo004{at}umn.edu.

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