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

Casadei, Cecilia M.; Gumiero, A.; Metcalfe, Clive; Murphy, Emma J.; Basran, Jaswir; Concilio, Maria Grazia; Teixeira, Susana; Schrader, Tobias E.; Fielding, Alistair J.; Ostermann, Andreas; Blakeley, Matthew P.; Raven, Emma Lloyd; Moody, P.C.E.

doi:10.1126/science.1254398

Cited by 138 publications

(194 citation statements)

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“…Our computational experiments indicate that the pK a substantially increases in CmpI. (B) Modified mechanism based on the observation that His52 in CCP CmpI is protonated in the neutron diffraction structure (8). Going from the initial peroxide complex to CmpI proceeds via two possible routes, one of which involves the Fe(IV)-OH intermediate.…”

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confidence: 99%

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

Chreifi

Baxter

Doukov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Fig. 1. Peroxidase mechanism. (A) Traditional "water-modified" mechanism of CmpI formation. In this mechanism, peroxide first coordinates with the heme iron, followed by proton transfer to the distal peroxide O atom via an ordered water molecule and the distal His. The protonation state of the distal His depends on the His pK a . Our computational experiments indicate that the pK a substantially increases in CmpI. (B) Modified mechanism based on the observation that His52 in CCP CmpI is protonated in the neutron diffraction structure (8). Going from the initial peroxide complex to CmpI proceeds via two possible routes, one of which involves the Fe(IV)-OH intermediate. (C) Mechanism of CmpII reduction, which includes a protoncoupled electron transfer event resulting in a net transfer of a proton from the distal His to the ferryl O atom.

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confidence: 99%

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

Chreifi

Baxter

Doukov

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…High electron density on the ferryl oxygen is thought to arise from the push effect of the trans-thiolate ligation (14,36). By contrast, HRP-II, cytochrome c peroxidase (37,38), and myoglobin (39), which have neutral histidines ligated to the heme, retain the ferryl oxo form. Resonance Raman measurements, X-ray absorption spectroscopy, Mössbauer spectroscopy, and X-ray crystallographic results suggest that the pK a is ≤ 4 for these ferryl states.…”

Section: Significancementioning

confidence: 99%

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Wang

Ullrich

Hofrichter

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pHjump spectrophotometry revealed a basic pK a of 10.0 for the Fe IV −O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10-10 7 M −1 ·s −1 . Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

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“…Macromolecular neutron crystallography (MNC) has been instrumental in answering longstanding questions about enzyme mechanisms [17][18][19] and ligand binding. [20,21] For example, neutron structures have revealed hydrated protons (or hydronium, H 3 O + ), which exist only transiently in bulk water, bound within proteins, [22][23][24] and have provided indisputable evidence of low-barrier hydrogen bonds in enzyme active sites.…”

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confidence: 99%

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

et al. 2016

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Proton transfer is integral to many biochemical processes. However, the direct observation and structural characterization of biological proton transfer has hitherto not been possible. Here, neutron crystallography directly locates two protons before and after a pH-induced two-proton transfer between the catalytic aspartic acid residues and the hydroxyl group of the bound clinical drug, darunavir, in the catalytic site of enzyme HIV-1 protease. The two-proton transfer is triggered by electrostatic effects arising from protonation state changes of surface residues far from the active site. The mechanism and pH effect are supported by QM/MM calculations. We propose that the low-pH proton configuration in the catalytic site is critical for the catalytic action of this enzyme and may apply more generally to other aspartic proteases. Neutrons therefore represent a superb probe to obtain structural details for proton transfer reactions in biological systems at a truly atomic level.

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

Cited by 138 publications

References 50 publications

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

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

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Long‐Range Electrostatics‐Induced Two‐Proton Transfer Captured by Neutron Crystallography in an Enzyme Catalytic Site

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