Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617.
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 (Iota) 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.
Correia
Cristina
C
REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
Besson
Stéphane
S
Brondino
Carlos D
CD
González
Pablo J
PJ
Fauque
Guy
G
Lampreia
Jorge
J
Moura
Isabel
I
Moura
José J G
JJ
D034241:Alteromonadaceae / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004586:Electrophoresis; D006150:Guanine Nucleotides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism; D050901:Nitrate Reductase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D011622:Pterins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D006150:Guanine Nucleotides; D011622:Pterins; C063761:molybdopterin guanine dinucleotide; D006418:Heme; D008982:Molybdenum; D007501:Iron; D050901:Nitrate Reductase