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Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study

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Abstract

In this article, we consider, in detail, the second half-cycle of the six-electron nitrite reduction mechanism catalyzed by cytochrome c nitrite reductase. In total, three electrons and four protons must be provided to reach the final product, ammonia, starting from the HNO intermediate. According to our results, the first event in this half-cycle is the reduction of the HNO intermediate, which is accomplished by two PCET reactions. Two isomeric radical intermediates, HNOH• and H2NO•, are formed. Both intermediates are readily transformed into hydroxylamine, most likely through intramolecular proton transfer from either Arg114 or His277. An extra proton must enter the active site of the enzyme to initiate heterolytic cleavage of the N–O bond. As a result of N–O bond cleavage, the H2N+ intermediate is formed. The latter readily picks up an electron, forming H2N+•, which in turn reacts with Tyr218. Interestingly, evidence for Tyr218 activity was provided by the mutational studies of Lukat (Biochemistry 47:2080, 2008), but this has never been observed in the initial stages of the overall reduction process. According to our results, an intramolecular reaction with Tyr218 in the final step of the nitrite reduction process leads directly to the final product, ammonia. Dissociation of the final product proceeds concomitantly with a change in spin state, which was also observed in the resonance Raman investigations of Martins et al. (J Phys Chem B 114:5563, 2010).

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Abbreviations

CcNiR:

Cytochrome c nitrite reductase

DFT:

Density functional theory

ET:

Electron transfer

MO:

Molecular orbitals

PCET:

Proton-coupled electron transfer

PES:

Potential energy surface

PT:

Proton transfer

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Acknowledgments

We gratefully acknowledge financial support of this work by the Max-Planck society. We thank Dr. Dimitrios Pantazis, Christopher Pollock, and Jessica Barilone for helpful comments on the manuscript.

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Authors and Affiliations

  1. Max-Planck Institut für Chemische Energiekonversion, Stiftstr. 34-36, 45470, Mülheim an der Ruhr, Germany

    Dmytro Bykov & Frank Neese

  2. Institut für Physikalische and Theoretische Chemie, University of Bonn, Wegelerstr. 12, 53115, Bonn, Germany

    Matthias Plog

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  1. Dmytro Bykov
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  2. Matthias Plog
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  3. Frank Neese
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Correspondence to Frank Neese.

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Bykov, D., Plog, M. & Neese, F. Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study. J Biol Inorg Chem 19, 97–112 (2014). https://doi.org/10.1007/s00775-013-1065-6

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