Publications

• Pathway for H2O2 and O2 detoxification in Clostridium acetobutylicum.

  Riebe O., Fischer R.J., Wampler D.A., Kurtz D.M. Jr., Bahl H.

  An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H2O2 and O2 detoxification p ... >> More

  An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H2O2 and O2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H2O2 oxidoreductase) and NADH oxidase (NADH : O2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H2O2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H2O2 and O2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase. << Less

  Microbiology 155:16-24(2009) [PubMed] [EuropePMC]

• NADH binding site and catalysis of NADH peroxidase.

  Stehle T., Claiborne A., Schulz G.E.

  The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure ... >> More

  The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and sulfenic acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation-sensitive sulfenic acid and propose a reaction mechanism. << Less

  Eur. J. Biochem. 211:221-226(1993) [PubMed] [EuropePMC]

• O2 and reactive oxygen species detoxification complex, composed of O2-responsive NADH:rubredoxin oxidoreductase-flavoprotein A2-desulfoferrodoxin operon enzymes, rubperoxin, and rubredoxin, in Clostridium acetobutylicum.

  Kawasaki S., Sakai Y., Takahashi T., Suzuki I., Niimura Y.

  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 uncharacte ... >> More

  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. << Less

  Appl. Environ. Microbiol. 75:1021-1029(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structure of the native cysteine-sulfenic acid redox center of enterococcal NADH peroxidase refined at 2.8-A resolution.

  Yeh J.I., Claiborne A., Hol W.G.J.

  In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resoluti ... >> More

  In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at -160 degrees C was applied. The structure of the native enzyme to 2.8 A resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms C alpha-C beta-S gamma-O roughly define a planar arrangement which is stacked parallel to the si face of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 A from FAD-C4A. His10-N epsilon 2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 A. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E.NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995) Biochemistry 34, 14114-14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction. << Less

  Biochemistry 35:9951-9957(1996) [PubMed] [EuropePMC]