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

• Type: Article in International Scientific Journal
• Date: 2005

Authors

Pinho, D (Author) Other; Besson, S (Author) Other; Silva, PJ (Author) Other; de Castro, B (Author) FCUP; Moura, I (Author) Other

Journal

Title: Biochimica et Biophysica Acta - General Subjects

Vol. 1723

Pages: 151-162

ISSN: 0304-4165

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 9 Citations

Scopus - 11 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-39H

DOI: 10.1016/j.bbagen.2005.02.008

Abstract (EN): 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(W) 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.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 12