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Applied and Environmental Microbiology, March 2005, p. 1522-1530, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1522-1530.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

Amy M. Grunden,1 Francis E. Jenney Jr.,2 Kesen Ma,2,{dagger} Mikyoung Ji,1 Michael V. Weinberg,2 and Michael W. W. Adams2*

Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia,2 Department of Microbiology, North Carolina State University, Raleigh, North Carolina1

Received 1 January 2004/ Accepted 19 October 2004

A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of –173 mV at 23°C (–193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Life Sciences Bldg., University of Georgia, Athens, GA 30602-7229. Phone: (706) 542-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.

{dagger} Present address: Department of Biology, University of Waterloo, Ontario N2L3GI, Canada.

Applied and Environmental Microbiology, March 2005, p. 1522-1530, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1522-1530.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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