Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

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Science  11 Jul 2014:
Vol. 345, Issue 6193, pp. 193-197
DOI: 10.1126/science.1254398

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Peroxidase proton placement

Heme enzymes catalyze a variety of biochemical oxidations through the activation of oxygen by iron. Casadei et al. used neutron crystallography to elucidate the mechanism of cytochrome c peroxidase (see the perspective by Groves and Boaz). In the highly reactive intermediate state termed compound I, the iron(IV) oxo, or ferryl, fragment was not protonated, whereas a nearby histidine residue was protonated. The sensitivity of neutron scattering to proton locations revealed these protonation states, where more common techniques, such as x-ray diffraction, have yielded more ambiguous results.

Science, this issue p. 193; see also p. 142


Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O–O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.

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