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Characterization of two distinct pyruvate ferredoxin oxidoreductases from hyperthermophilic organisms
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Abstracts
GO17 CHARACTERIZATION
OF TWO DISTINCT PYRUVATE FERREDOXIN OXIDOREDUCTASES FROM HYPERTHERMOPHILIC ORGANISMS
Eugene T. Smith, Jenny M. Blarney and Michael W. W. Adams Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 Hyperthermophiles are a recently discovered group of microorganisms that grow at temperatures near 100°C. To date most of the biochemical studies on this group have been carried out with the archaeon (formerly archaebacterium), Pyrococcus furiosus (Pf), which grows optimally at 100°C, and the (eu)bacterium, Thermotoga maritima (Tm), which grows up to 90°C (1). Both are strictly anaerobic heterotrophs that grow by the fermentation of carbohydrates. From these two organisms we have purified a key enzyme in their fermentative pathways, pyruvate ferredoxin oxidoreductase (POR: Ref. 2). POR catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and CO2 using ferredoxin as the electron carrier. Both enzymes are obligately dependent upon CoASH for activity and are active at temperatures above 95OCwith virtually no activity at 25OC. Pf POR and Tm POR are each trimeric proteins (spy) with molecular weights of about 100,000. Interestingly, this is half the size of PORs that have been purified from mesophilic anaerobes (2). They are both inactivated by 02 with half-lives in air of about 15 min. Each contains a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] clusters. However, the two enzymes differ in that Pf POR also contains 1 Cu and 1 Zn atom/molecule whereas in Tm POR the only detectable metal is Fe. Oxidized Pf POR exhibited an EPR signal characteristic of a Cu2+ center. The addition of pyruvate to the enzyme increased the intensity of the Cu-derived EPR signal and also caused the formation of a radical species. The latter was assigned to a hydroxyethyl-TPP radical intermediate as its EPR signal became measurably broader when the enzyme was treated with 2-13C-pyruvate. In contrast, pyruvate had no effect on the EPR properties of oxidized Tm POR. Furthermore, the addition of CoASH to oxidized Pf POR caused the reduction of the Cu2+ center, while in Tm POR a novel FeS center was reduced. The addition of both substrates, CoASH and pyruvate, to either enzyme resulted in the reduction of their [4Fe-4S] clusters. These two enzymes therefore appear to have distinctly different means of catalyzing the oxidation of pyruvate at extreme temperatures. Potential catalytic mechanisms will be presented in which a Cu site in Pf POR and an FeS center in Tm POR function in acetyl transfer. 1. Adams, M. W. W. Advs. Inorg. Chem. 38,341 (1992) 2. Blarney, J. M. and Adams, M. W. W. Biochim. Biophys. Acta 1161,19 (1993)
Abstracts
GO17 CHARACTERIZATION
OF TWO DISTINCT PYRUVATE FERREDOXIN OXIDOREDUCTASES FROM HYPERTHERMOPHILIC ORGANISMS
Eugene T. Smith, Jenny M. Blarney and Michael W. W. Adams Department of Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602 Hyperthermophiles are a recently discovered group of microorganisms that grow at temperatures near 100°C. To date most of the biochemical studies on this group have been carried out with the archaeon (formerly archaebacterium), Pyrococcus furiosus (Pf), which grows optimally at 100°C, and the (eu)bacterium, Thermotoga maritima (Tm), which grows up to 90°C (1). Both are strictly anaerobic heterotrophs that grow by the fermentation of carbohydrates. From these two organisms we have purified a key enzyme in their fermentative pathways, pyruvate ferredoxin oxidoreductase (POR: Ref. 2). POR catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and CO2 using ferredoxin as the electron carrier. Both enzymes are obligately dependent upon CoASH for activity and are active at temperatures above 95OCwith virtually no activity at 25OC. Pf POR and Tm POR are each trimeric proteins (spy) with molecular weights of about 100,000. Interestingly, this is half the size of PORs that have been purified from mesophilic anaerobes (2). They are both inactivated by 02 with half-lives in air of about 15 min. Each contains a thiamine pyrophosphate (TPP) cofactor and at least two [4Fe-4S] clusters. However, the two enzymes differ in that Pf POR also contains 1 Cu and 1 Zn atom/molecule whereas in Tm POR the only detectable metal is Fe. Oxidized Pf POR exhibited an EPR signal characteristic of a Cu2+ center. The addition of pyruvate to the enzyme increased the intensity of the Cu-derived EPR signal and also caused the formation of a radical species. The latter was assigned to a hydroxyethyl-TPP radical intermediate as its EPR signal became measurably broader when the enzyme was treated with 2-13C-pyruvate. In contrast, pyruvate had no effect on the EPR properties of oxidized Tm POR. Furthermore, the addition of CoASH to oxidized Pf POR caused the reduction of the Cu2+ center, while in Tm POR a novel FeS center was reduced. The addition of both substrates, CoASH and pyruvate, to either enzyme resulted in the reduction of their [4Fe-4S] clusters. These two enzymes therefore appear to have distinctly different means of catalyzing the oxidation of pyruvate at extreme temperatures. Potential catalytic mechanisms will be presented in which a Cu site in Pf POR and an FeS center in Tm POR function in acetyl transfer. 1. Adams, M. W. W. Advs. Inorg. Chem. 38,341 (1992) 2. Blarney, J. M. and Adams, M. W. W. Biochim. Biophys. Acta 1161,19 (1993)