Isolation and spectroscopic characterization of the membrane-bound nitrate reductase from Pseudomonas chlororaphis DSM 50135.
A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM 50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as alpha, beta and gamma, respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. The isolated enzyme possesses several iron-sulfur clusters and a molybdopterin guanine dinucleotide active center. The iron-sulfur clusters can be grouped in two classes according to their redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy. The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an -OHx ligand. Dithionite reduces the Mo(V) center to Mo(IV) and subsequent reoxidization with nitrate originates a new Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes.
Pinho
Dora
D
REQUIMTE, Departamento de Química, Faculdade de Ciências da Universidade do Porto, Rua Campo Alegre 687, 4169-007 Porto, Portugal.
Besson
Stéphane
S
Silva
Pedro J
PJ
de Castro
Baltazar
B
Moura
Isabel
I
D002462:Cell Membrane / Q000201:enzymology; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry / Q000302:isolation & purification*; D011549:Pseudomonas / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D009565:Nitrate Reductases; D050901:Nitrate Reductase