Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 24, Pages 8797-8802Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1408115111
Keywords
X-ray absorption spectroscopy; DFT; dioxygen activation; biofuels
Categories
Funding
- National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health [R01DK031450, NIH P41GM103393]
- Biotechnology and Biological Sciences Research Council [BB/I014802/1, BB/L000423/1]
- John Stauffer Stanford Graduate Fellowship
- U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
- Department of Energy Office of Biological and Environmental Research
- National Institutes of Health, National Institute of General Medical Sciences [P41GM103393]
- Biotechnology and Biological Sciences Research Council [BB/L000423/1, BB/L021633/1] Funding Source: researchfish
- BBSRC [BB/L000423/1, BB/L021633/1] Funding Source: UKRI
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Strategies for O-2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O-2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O-2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O-2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O-2 binding with very little reorganization energy.
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