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Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

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Abstract

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]+ cluster and overlapping signals associated with at least three types of [4Fe–4S]+ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases.

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Abbreviations

Bis–Tris:

[Bis(2-hydroxyethyl)imino]tris(hydroxymethyl)methane

Bis–Tris propane:

1,3-Bis[tris(hydroxymethyl)methylamino]propane

DDM:

n-Dodecyl-β-d-maltoside

DMSO:

Dimethyl sulfoxide

EPR:

Electron paramagnetic resonance

MGD:

Molybdopterin guanine dinucleotide

Nar:

Respiratory nitrate reductase

PA:

Phenethyl alcohol

PAGE:

Polyacrylamide gel electrophoresis

SDS:

Sodium dodecyl sulfate

Tris–HCl:

Tris(hydroxymethyl)aminomethane hydrochloride

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Acknowledgments

C.C. and P.J.G. (SFRH/BPD/29812/2006) thank FCT for a fellowship grant. C.D.B. and J.J.G.M. thank SECYT (Argentina) and GRICES (Portugal) for a binational grant. We thank M. Bauzan (LCB-CNRS, Marseille, France) for growing the bacteria used in the present study. This work was supported by project POCI/QUI/57641/2004 in Portugal and SEPCYT:PICT 2003-06-13872, CONICET PIP 5370, and CAI + D − UNL in Argentina. C.D.B. is a member of CONICET-Argentina.

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Authors and Affiliations

  1. REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516, Caparica, Portugal

    Cristina Correia, Stéphane Besson, Pablo J. González, Jorge Lampreia, Isabel Moura & José J. G. Moura

  2. UIBD, Faculdade de Engenharias e Ciências Naturais, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024, Lisbon, Portugal

    Stéphane Besson

  3. Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000, Santa Fe, Argentina

    Carlos D. Brondino

  4. Laboratoire de Microbiologie, Institut de Recherche Pour le Développement, UR 101, IFR-BAIM, Universités de Provence et de la Méditerranée, ESIL, Case 925, 163 Avenue de Luminy, 13288, Marseille Cedex 9, France

    Guy Fauque

  5. Laboratoire de Microbiologie, Géochimie et Ecologie Marines, CNRS UMR 6117, Campus de Luminy, Case 901, 13288, Marseille Cedex 09, France

    Guy Fauque

Authors
  1. Cristina Correia
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  2. Stéphane Besson
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  4. Pablo J. González
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  5. Guy Fauque
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  6. Jorge Lampreia
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  7. Isabel Moura
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  8. José J. G. Moura
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Corresponding authors

Correspondence to Carlos D. Brondino or José J. G. Moura.

Electronic supplementary material

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775_2008_416_MOESM1_ESM.pdf

Mo(V) EPR signal of dithionite reduced Ma. hydrocarbonoclasticus 617 Nar at pH 6 after 45 minutes of nitrate addition (1) and spectrum obtained after reoxidizing the sample of spectrum 1 with air (2). Experimental conditions are the same of Figure 7 (PDF 29 kb)

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Correia, C., Besson, S., Brondino, C.D. et al. Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617. J Biol Inorg Chem 13, 1321–1333 (2008). https://doi.org/10.1007/s00775-008-0416-1

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