Isolation and spectroscopic characterization of the membrane-bound nitrate reductase from Pseudomonas chlororaphis DSM 50135

• Published in: Biochimica et biophysica acta Vol. 1723; no. 1; pp. 151 - 162
• Main Authors: Pinho, Dora, Besson, Stéphane, Silva, Pedro J., de Castro, Baltazar, Moura, Isabel
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 25.05.2005
• Subjects: Cell Membrane - enzymology; Electrochemistry; Electron Spin Resonance Spectroscopy; EPR; Heme c; Iron–sulfur centers; Molybdenum; Nitrate Reductase; Nitrate Reductases - chemistry; Nitrate Reductases - isolation & purification; Pseudomonas - enzymology; Spectrophotometry, Ultraviolet; Nitrate reductase; Molybdenum; EPR; Heme c; Iron–sulfur centers
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2005.02.008

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 α, β and γ, 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 –OH x 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.