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

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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.

Original languageEnglish
Pages (from-to)1321-1333
Number of pages13
JournalJournal of Biological Inorganic Chemistry
Volume13
Issue number8
DOIs
Publication statusPublished - 1 Nov 2008

Fingerprint

Marinobacter
Nitrate Reductase
Heme
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Membranes
Dithionite
Enzymes
Nitrates
Nitrate Reductases
Molecules
Molecular mass
Nitrites
Protons
Spectrum Analysis
Temperature

Keywords

  • Denitrification
  • Electron paramagnetic resonance
  • Marinobacter hydrocarbonoclasticus
  • Molybdenum
  • Nitrate reductase

Cite this

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title = "Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617",
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.",
keywords = "Denitrification, Electron paramagnetic resonance, Marinobacter hydrocarbonoclasticus, Molybdenum, Nitrate reductase",
author = "Cristina Correia and St{\'e}phane Besson and Brondino, {Carlos D.} and Gonz{\'a}lez, {Pablo J.} and Guy Fauque and Jorge Lampreia and Isabel Moura and Moura, {Jos{\'e} J. G.}",
note = "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.",
year = "2008",
month = "11",
day = "1",
doi = "10.1007/s00775-008-0416-1",
language = "English",
volume = "13",
pages = "1321--1333",
journal = "Journal of Biological Inorganic Chemistry",
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TY - JOUR

T1 - Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

AU - Correia, Cristina

AU - Besson, Stéphane

AU - Brondino, Carlos D.

AU - González, Pablo J.

AU - Fauque, Guy

AU - Lampreia, Jorge

AU - Moura, Isabel

AU - Moura, José J. G.

N1 - 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.

PY - 2008/11/1

Y1 - 2008/11/1

N2 - 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.

AB - 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.

KW - Denitrification

KW - Electron paramagnetic resonance

KW - Marinobacter hydrocarbonoclasticus

KW - Molybdenum

KW - Nitrate reductase

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U2 - 10.1007/s00775-008-0416-1

DO - 10.1007/s00775-008-0416-1

M3 - Article

VL - 13

SP - 1321

EP - 1333

JO - Journal of Biological Inorganic Chemistry

JF - Journal of Biological Inorganic Chemistry

SN - 1432-1327

IS - 8

ER -