Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 113, Issue 5, Pages 1226-1231Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1521664113
Keywords
compound I; peroxidase; femtosecond crystallography; XFEL
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Funding
- NIH [GM57353]
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
- Department of Energy Office of Biological and Environmental Research
- NIH, National Institute of General Medical Sciences [P41GM103393]
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The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray-generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-angstrom crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe (IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)OH. We also find that the pK(a) of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.
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