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Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs

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Abstract

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V max and the nitrite dependence of the catalytic current–potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.

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Abbreviations

EcNrfA:

Escherichia coli NrfA

Hepes:

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

LMCT:

Ligand-to-metal charge transfer

MCD:

Magnetic circular dichroism

MOTTLE:

Magnetic circular dichroism compatible optically transparent thin layer electrochemistry

PFE:

Protein-film electrochemistry

PGE:

Pyrolytic graphite edge

SHE:

Standard hydrogen electrode

TvNiR:

Thioalkalivibrio nitratireducens octaheme nitrite reductase

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Acknowledgments

We are grateful to James Durrant and Li Xiaoe (Imperial College London) for providing the SnO2 electrodes used in this work and to the reviewers for their insights into the interpretation of the data presented. The work was funded by the UK Biotechnology and Biosciences Research Council through grants B15211, C007808, B18695, G024758, and G009228.

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Authors and Affiliations

  1. Centre for Molecular and Structural Biochemistry, School of Chemistry and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK

    Rose-Marie A. S. Doyle, Sophie J. Marritt, James D. Gwyer, Thomas G. Lowe, Myles R. Cheesman & Julea N. Butt

  2. A.N. Bakh Institute of Biochemistry RAS, Leninsky pr.33, 119071, Moscow, Russia

    Tamara V. Tikhonova & Vladimir O. Popov

  3. NBICS Centre of Russian National Research Centre “Kurchatov Institute”, Akademika Kurchatova sq. 1, Moscow, 123182, Russia

    Vladimir O. Popov

Authors
  1. Rose-Marie A. S. Doyle
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  2. Sophie J. Marritt
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  3. James D. Gwyer
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  4. Thomas G. Lowe
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  5. Tamara V. Tikhonova
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  6. Vladimir O. Popov
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  7. Myles R. Cheesman
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  8. Julea N. Butt
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Correspondence to Julea N. Butt.

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Doyle, RM.A.S., Marritt, S.J., Gwyer, J.D. et al. Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs. J Biol Inorg Chem 18, 655–667 (2013). https://doi.org/10.1007/s00775-013-1011-7

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  • DOI: https://doi.org/10.1007/s00775-013-1011-7

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