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Abstract

, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit), showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit) and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ≥10 and ≥95 °C, respectively. At pH 7 and 83 °C, apparent values for NADH and O were 3 μM and 1.9 mM, respectively, and the specific activity at 1.4 mM O was 60 μmol min mg; 62 % of NADH-derived reducing equivalents were recovered as HO and the rest probably generated HO. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5′-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high , O is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

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2009-01-01
2019-11-22
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