JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 8, pp 1321–1333

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

  • Cristina Correia
  • Stéphane Besson
  • Carlos D. Brondino
  • Pablo J. González
  • Guy Fauque
  • Jorge Lampreia
  • Isabel Moura
  • José J. G. Moura
Original Paper

DOI: 10.1007/s00775-008-0416-1

Cite this article as:
Correia, C., Besson, S., Brondino, C.D. et al. J Biol Inorg Chem (2008) 13: 1321. doi:10.1007/s00775-008-0416-1

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 Em = +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.

Keywords

Nitrate reductase Electron paramagnetic resonance Molybdenum Denitrification Marinobacter hydrocarbonoclasticus 

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

Supplementary material

775_2008_416_MOESM1_ESM.pdf (29 kb)
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)

Copyright information

© SBIC 2008

Authors and Affiliations

  • Cristina Correia
    • 1
  • Stéphane Besson
    • 1
    • 2
  • Carlos D. Brondino
    • 3
  • Pablo J. González
    • 1
  • Guy Fauque
    • 4
    • 5
  • Jorge Lampreia
    • 1
  • Isabel Moura
    • 1
  • José J. G. Moura
    • 1
  1. 1.REQUIMTE/CQFB, Departamento de QuímicaFaculdade de Ciências e Tecnologia da Universidade Nova de LisboaCaparicaPortugal
  2. 2.UIBD, Faculdade de Engenharias e Ciências NaturaisUniversidade Lusófona de Humanidades e TecnologiasLisbonPortugal
  3. 3.Facultad de Bioquímica y Ciencias BiológicasUniversidad Nacional del LitoralSanta FeArgentina
  4. 4.Laboratoire de Microbiologie, Institut de Recherche Pour le Développement, UR 101, IFR-BAIMUniversités de Provence et de la Méditerranée, ESILMarseille Cedex 9France
  5. 5.Laboratoire de MicrobiologieGéochimie et Ecologie Marines, CNRS UMR 6117Marseille Cedex 09France