Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer

Georges Chreifi^a,¹, Elizabeth L. Baxter^b,¹,Tzanko Doukov^b,Aina E. Cohen^b,Scott E. McPhillips^b,Jinhu Song^b,Yergalem T. Meharenna^a,S. Michael Soltis^b,², andThomas L. Poulos^a,^c,^d,²

^aDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;

^bMacromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;

^cDepartment of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3900;

^dDepartment of Chemistry, University of California, Irvine, CA 92697-3900 

 PNAS, 2016, 113, 1226

Abstract

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-Å 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. 

 

 

 X線自由電子レーザーを用いて、cytochrome c peroxidase(CCP)のFe(IV)=O種の結晶構造を決定し、Fe(IV)=Oから、Fe(III)-OHになる過程で、His52からプロトンが水を介して移動することを示したと報告されています。

なお、通常のX線で測定すると、鉄中心が還元され、Fe-O間の結合が伸びたものしかと報告がなくオキソ種ではなく、ヒドロキソ種であると言われていました。

http://www.pnas.org/content/113/5/1226.full.pdf