O 2 and Reactive Oxygen Species Detoxification Complex, Composed of O 2 -Responsive NADH:Rubredoxin Oxidoreductase-Flavoprotein A2-Desulfoferrodoxin Operon Enzymes, Rubperoxin, and Rubredoxin, in Clostridium acetobutylicum

Abstract

Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O(2)-flow culture conditions, where the cells consume O(2) proficiently. An O(2)-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O(2) stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H(2)O(2)-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H(2)O-forming NADH oxidase with a high affinity for O(2) (the K(m) for O(2) is 2.9 +/- 0.4 microM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O(2)(-) reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H(2)O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O(2) and a high affinity for H(2)O(2) (the K(m)s for O(2) and H(2)O(2) are 303 +/- 39 microM and <or=1 microM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O(2). Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O(2), O(2)(-), and H(2)O(2) by using an O(2)-responsive rubredoxin as a common electron carrier protein.