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Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

Christopher L. Case^1,

¹ Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
² Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA
³ Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

Correspondence
Biswarup Mukhopadhyay
biswarup{at}vt.edu

Methanocaldococcus jannaschii, 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 Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)^–1, showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)^–1 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 K_m values for NADH and O₂ were 3 µM and 1.9 mM, respectively, and the specific activity at 1.4 mM O₂ was 60 µmol min^–1 mg^–1; 62 % of NADH-derived reducing equivalents were recovered as H₂O₂ and the rest probably generated H₂O. 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 K_m, O₂ is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

Present address: Yale University School of Medicine, Section of Microbial Pathogenesis, 295 Congress Avenue, BCMM 345, New Haven, CT 06536, USA.

A supplementary table describing the purification of rMjNox is available with the online version of this paper.