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NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O2 reactivity

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Abstract

Iron(II)-containing homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of homoprotocatechuate (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron or electrons from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD–HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII–O2 ·− species that is not observed during turnover of the wild-type FeHPCD. The factors governing formation of the MIII–O2 ·− species are explored here by EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and the dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD–4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD–ligand complexes that bind NO are also active in normal O2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from the ability of the enzyme to maintain the approximately 0.8-V difference in the solution redox potentials of FeII and MnII. Owing to the higher potential of Mn, the formation of the NO adduct or the O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active-site His200. The same nonoptimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover. Accordingly, past studies have shown that intermediate FeIII species are observed for these mutant enzymes.

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Abbreviations

4NC:

4-Nitrocatechol

DEA NONOate:

Diethylamine NONOate diethylammonium salt

DHM:

Dihydroxymandelic acid

EPR:

Electron paramagnetic resonance

FeHPCD:

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum

HPCA:

Homoprotocatechuate

HPCD:

Homoprotocatechuate 2,3-dioxygenase

ICP-AES:

Inductively coupled plasma atomic emission spectroscopy

MnHPCD:

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum in which the FeII is replaced by MnII

RFQ:

Rapid freeze quench

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Acknowledgments

This work was supported by grants GM24689 (to J.D.L), GM33162 (to L.Q.), and GM77387 (to M.P.H.).

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Authors and Affiliations

  1. Department of Chemistry, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA, 15213, USA

    Joshua A. Hayden & Michael P. Hendrich

  2. Department of Chemistry, University of Minnesota, Minneapolis, MN, 55455, USA

    Erik R. Farquhar & Lawrence Que

  3. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA

    John D. Lipscomb

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  1. Joshua A. Hayden
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  2. Erik R. Farquhar
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  3. Lawrence Que
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  4. John D. Lipscomb
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  5. Michael P. Hendrich
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Corresponding author

Correspondence to Michael P. Hendrich.

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Hayden, J.A., Farquhar, E.R., Que, L. et al. NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O2 reactivity. J Biol Inorg Chem 18, 717–728 (2013). https://doi.org/10.1007/s00775-013-1016-2

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  • DOI: https://doi.org/10.1007/s00775-013-1016-2

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