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

Abstract

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 6C, respectively. At pH 7 and 83 6C, apparent K m values for NADH and O 2 were 3 mM and 1.9 mM, respectively, and the specific activity at 1.4 mM O 2 was 60 mmol min "1 mg "1 ; 62 % of NADH-derived reducing equivalents were recovered as H 2 O 2 and the rest probably generated H 2 O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,59-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m , O 2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

Characterization of NADH Oxidase/NADPH Polysulfide Oxidoreductase and Its Unexpected Participation in Oxygen Sensitivity in an Anaerobic Hyperthermophilic Archaeon

“…In addition to these enzymes, a heterodimeric NADH oxidase from T. maritima, the ␣ subunit of which is a NOX homolog, has been reported to have quite high activity (144 U/mg) (42). The solution of purified rTK1481 was yellowish, suggesting the presence of oxidized prosthetic FAD groups, which have also been observed in other NOXs from hyperthermophiles (4,10,14,17,28,38,41,42) in this enzyme. This was supported by the presence of absorption peaks at 450 nm in the spectrum of the purified enzyme (Fig.…”

“…In this assay, the initial rates of both NAD(P)H oxidation and H 2 O 2 formation were measured to determine the stoichiometry of the reaction catalyzed by this enzyme. As shown in Table 2, rTK1481 actually showed both (4,11,14,17,38). In addition to these enzymes, a heterodimeric NADH oxidase from T. maritima, the ␣ subunit of which is a NOX homolog, has been reported to have quite high activity (144 U/mg) (42).…”

“…One possible reason for this phenomenon may be the generation of excess H 2 O 2 accompanied by the removal of dissolved oxygen by the function of TK1481 in the sulfur-free medium. In several anaerobic hyperthermophiles, including members of the genera Thermococcus and Pyrococcus, superoxide reductase has been proposed to convert the highly toxic superoxide to H 2 O 2 (10), and many NOX homologs also produce H 2 O 2 from dissolved oxygen (4,17,28,38,41,42). Since these hyperthermophiles do not encode catalase in their genomes, the moderately toxic stressor H 2 O 2 is thought to be reductively removed by proteins with peroxidase activity (25,27,39).…”

“…Likewise, the active Cys43 residue in the CoADR from Staphylococcus aureus forms a Cys43-SSCoA redox center during turnover (23). The NOX homologs in hyperthermophiles are considered to play important roles in the defense against oxidative stress (4,14,17,28,38,41,42). However, the actual physiological functions of NOXs in anaerobic hyperthermophiles have not yet been completely elucidated, mainly due to a lack of useful genetic manipulation systems for these organisms.…”

“…(38), Archaeoglobus fulgidus (17,28), Methanocaldococcus jannaschii (4), and Thermotoga spp. (41,42).…”