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• W2046287787 abstract "Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO2 fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V max = 1.6 unit/mg (k cat = 3.2 s−1), KmAcetyl-CoA = 9 μm, and KmCO2 = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of itsk cat/K m values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis. Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2. The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO2 fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V max = 1.6 unit/mg (k cat = 3.2 s−1), KmAcetyl-CoA = 9 μm, and KmCO2 = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of itsk cat/K m values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis. pyruvate:ferredoxin oxidoreductase CO dehydrogenase 3-(N-morpholino)-propanesulfonate acetyl-CoA synthase lactate dehydrogenase 4-morpholineethanesulfonic acid high pressure liquid chromatography ferredoxin Organisms from all three kingdoms of life (Bacteria, Archaea, and Eukarya) metabolize pyruvate in biosynthetic and catabolic reactions. Mitochondria and aerobic bacteria couple the oxidative decarboxylation of pyruvate to the reduction of NAD+ by the pyruvate dehydrogenase multienzyme complex (1Patel M.S. Roche T.E. FASEB J. 1990; 4: 3224-3233Crossref PubMed Scopus (502) Google Scholar). In many anaerobic organisms, pyruvate:ferredoxin oxidoreductase (PFOR)1 catalyzes the oxidative decarboxylation of pyruvate to CO2 and acetyl-CoA (Reaction 1) (2Menon S. Ragsdale S.W. Biochemistry. 1997; 36: 8484-8494Crossref PubMed Scopus (62) Google Scholar, 3Hrdý I. Müller M. J. Mol. Evol. 1995; 41: 388-396Crossref PubMed Scopus (0) Google Scholar, 4Neuer G. Bothe H. Biochim. Biophys. Acta. 1982; 716: 358-365Crossref PubMed Scopus (39) Google Scholar, 5Yakunin A.F. Hallenbeck P.C. Biochim. Biophys. Acta. 1998; 1409: 39-49Crossref PubMed Scopus (43) Google Scholar, 6Adams M.W.W. Kletzin A. Adv. Protein Chem. 1996; 48: 101-180Crossref PubMed Google Scholar). In anaerobic acetogenic bacteria, 2 moles of CO2 generated from the decarboxylation of 2 moles of pyruvate are reduced to another mol of acetyl-CoA in an autotrophic biosynthetic scheme known as the Wood-Ljungdahl pathway (7Ragsdale S.W. Kumar M. Zhao S. Menon S. Seravalli J. Doukov T. Krautler B. Vitamin B12 and B12-Proteins. Wiley-VCH, Weinheim, Germany1998: 167-177Google Scholar, 8Ragsdale S.W. Biofactors. 1997; 9: 1-9Google Scholar, 9Ragsdale, S. W. (2000) in Biological Inorganic Chemistry: Structure and Reactivity (Valentine, J. S., Bertini, I., and Gray, H., eds), in press, University Science Books.Google Scholar). Thus, PFOR links glycolysis to the Wood-Ljungdahl pathway. The reverse reaction, carboxylation of acetyl-CoA, is an important reaction for anaerobes like methanogens and acetogens that fix CO2 by the Wood-Ljungdahl pathway (10Simpson P.G. Whitman W.B. Ferry J.G. Methanogenesis: Ecology Physiology, Biochemistry & Genetics. Chapman & Hall, London1993: 445-472Crossref Google Scholar, 11Shieh J.S. Whitman W.B. J. Bacteriol. 1987; 169: 5327-5329Crossref PubMed Google Scholar, 12Lapado J. Whitman W.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5598-5602Crossref PubMed Scopus (60) Google Scholar, 13Fuchs G. FEMS Microbiol. Rev. 1986; 39: 181-213Crossref Google Scholar). In this case, PFOR (pyruvate synthase) links the Wood-Ljungdahl pathway to the incomplete reductive tricarboxylic acid cycle, which generates biosynthetic intermediates.Pyruvate+CoASH→Acetyl­SCoA+CO2+2H++2e−REACTION 1 CO+H2O→CO2+2H++2e−REACTION 2 PFORs can be homo- (14Wahl R.C. Orme-Johnson W.H. J. Biol. Chem. 1987; 262: 10489-10496Abstract Full Text PDF PubMed Google Scholar) or heterodimeric (15Kerscher L. Oesterhelt D. Trends. Biochem. Sci. 1982; 7: 371-374Abstract Full Text PDF Scopus (117) Google Scholar) or heterotetrameric (6Adams M.W.W. Kletzin A. Adv. Protein Chem. 1996; 48: 101-180Crossref PubMed Google Scholar). It is thought that all PFORs evolved by rearrangements and fusions of four ancestral genes (16Kletzin A. Adams M.W.W. J. Bacteriol. 1996; 178: 248-257Crossref PubMed Google Scholar, 17Zhang Q. Iwasaki T. Wakagi T. Oshima T. J. Biochem. (Tokyo). 1996; 120: 587-599Crossref PubMed Scopus (80) Google Scholar). They contain 1–3 iron-sulfur clusters and thiamine pyrophosphate as prosthetic groups (15Kerscher L. Oesterhelt D. Trends. Biochem. Sci. 1982; 7: 371-374Abstract Full Text PDF Scopus (117) Google Scholar,18Bock A.K. Schonheit P. Teixeira M. FEBS Lett. 1997; 414: 209-212Crossref PubMed Scopus (16) Google Scholar, 19Menon A.L. Hendrix H. Hutchins A. Verhagen M. Adams M.W.W. Biochemistry. 1998; 37: 12838-12846Crossref PubMed Scopus (20) Google Scholar). The PFOR from Clostridium thermoaceticum is a 240-kDa homodimer, with two [4Fe-4S]2+/1+ clusters and one thiamin pyrophosphate/subunit (2Menon S. Ragsdale S.W. Biochemistry. 1997; 36: 8484-8494Crossref PubMed Scopus (62) Google Scholar, 14Wahl R.C. Orme-Johnson W.H. J. Biol. Chem. 1987; 262: 10489-10496Abstract Full Text PDF PubMed Google Scholar) The two electrons generated by the oxidative decarboxylation of pyruvate are transferred to an 8-iron ferredoxin (or possibly other electron carriers, see below) that in turn can reduce a variety of cellular enzymes. This electron pair can also be transferred directly to CODH, which reduces CO2 to CO, an intermediate in the Wood-Ljungdahl pathway (20Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google Scholar). The PFOR reaction has been most extensively studied in the forward (oxidative decarboxylation) direction beginning with a series of seminal studies published in 1971 (21Uyeda K. Rabinowitz J.C. J. Biol. Chem. 1971; 246: 3120-3125Abstract Full Text PDF PubMed Google Scholar, 22Uyeda K. Rabinowitz J.C. J. Biol. Chem. 1971; 246: 3111-3119Abstract Full Text PDF PubMed Google Scholar, 23Raeburn S. Rabinowitz J.C. Arch. Biochem. Biophys. 1971; 146: 9-20Crossref PubMed Scopus (32) Google Scholar, 24Raeburn S. Rabinowitz J.C. Arch. Biochem. Biophys. 1971; 146: 21-33Crossref PubMed Scopus (37) Google Scholar). Rabinowitz and co-workers (21Uyeda K. Rabinowitz J.C. J. Biol. Chem. 1971; 246: 3120-3125Abstract Full Text PDF PubMed Google Scholar, 22Uyeda K. Rabinowitz J.C. J. Biol. Chem. 1971; 246: 3111-3119Abstract Full Text PDF PubMed Google Scholar, 23Raeburn S. Rabinowitz J.C. Arch. Biochem. Biophys. 1971; 146: 9-20Crossref PubMed Scopus (32) Google Scholar, 24Raeburn S. Rabinowitz J.C. Arch. Biochem. Biophys. 1971; 146: 21-33Crossref PubMed Scopus (37) Google Scholar) isolated and characterized pyruvate:ferredoxin oxidoreductase. They also demonstrated that low potential electron donors, like reduced ferredoxin, can drive the reductive carboxylation of acetyl-CoA (24Raeburn S. Rabinowitz J.C. Arch. Biochem. Biophys. 1971; 146: 21-33Crossref PubMed Scopus (37) Google Scholar). 2At that time it was not clear whether acetyl-CoA was the substrate, so they used acetyl-phosphate in the presence of CoA and phosphotransacetylase. Subsequently, few studies of the pyruvate synthase activity have been published. Can PFOR also serve as a pyruvate synthase? A PFOR has been isolated from the methanogenic archaea, Methanosarcina barkeri (18Bock A.K. Schonheit P. Teixeira M. FEBS Lett. 1997; 414: 209-212Crossref PubMed Scopus (16) Google Scholar) and Methanobacterium thermoautotrophicum (25Tersteegen A. Linder D. Thauer R.K. Hedderich R. Eur. J. Biochem. 1997; 244: 862-868Crossref PubMed Scopus (61) Google Scholar). These enzymes must function in anabolic reactions, because methanogens cannot grow on substrates with a more complex structure than acetate. The M. barkeri enzyme was shown to catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA and the reductive carboxylation of acetyl-CoA with ferredoxin as an electron carrier (26Bock A.K. Kunow J. Glasemacher J. Schonheit P. Eur. J. Biochem. 1996; 237: 35-44Crossref PubMed Scopus (37) Google Scholar). The sequences of the methanogenic enzymes are closely related to those of the PFORs fromPyrococus furiosus and Thermotoga maritima (25Tersteegen A. Linder D. Thauer R.K. Hedderich R. Eur. J. Biochem. 1997; 244: 862-868Crossref PubMed Scopus (61) Google Scholar,26Bock A.K. Kunow J. Glasemacher J. Schonheit P. Eur. J. Biochem. 1996; 237: 35-44Crossref PubMed Scopus (37) Google Scholar), which function in a catabolic direction (27Blamey J.M. Adams M.W.W. Biochim. Biophys. Acta. 1993; 1161: 19-27Crossref PubMed Scopus (142) Google Scholar). Thus, these combined studies indicate that the same enzyme (PFOR) functions physiologically in either direction. This conclusion is supported by the finding that under some conditions, methanogens can grow, albeit poorly, on pyruvate (28Rajagopal B.S. LeGall J. Curr. Microbiol. 1994; 28: 307-311Crossref Scopus (9) Google Scholar, 29Bock A.-K. Schönheit P. J. Bacteriol. 1995; 177: 2002-2007Crossref PubMed Google Scholar). Yoon et al. (37Yoon K.S. Hille R. Hemann C. Tabita F.R. J. Biol. Chem. 1999; 274: 29772-29778Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) have also studied the pyruvate synthase reaction of the PFOR from Chloribium tepidum. The results described above support the hypothesis that PFOR and pyruvate synthase are the same enzyme. However, the reverse reaction of PFOR has been scantily studied and several examples exist of distinct enzymes catalyzing forward or reverse reactions. For example, fumarate reductase and succinate dehydrogenase preferentially catalyze opposing reactions (30Hirst J. Sucheta A. Ackrell B.A.C. Armstrong F.A. J. Am. Chem. Soc. 1996; 118: 5031-5038Crossref Scopus (105) Google Scholar, 31Hirst J. Ackrell B.A.C. Armstrong F.A. J. Am. Chem. Soc. 1997; 119: 7434-7439Crossref Scopus (40) Google Scholar) and are separately regulated and distinct gene products (32Maklashina E. Berthold D.A. Cecchini G. J. Bacteriol. 1998; 180: 5989-5996Crossref PubMed Google Scholar, 33Hirsch C.A. Rasminsky M. Davis B.D. Lin E.C.C. J. Biol. Chem. 1963; 238: 3770-3774Abstract Full Text PDF PubMed Google Scholar). In many organisms, H+ reduction and H2 oxidation are catalyzed by separate enzymes with the converse catalytic biases (34Pershad H.R. Duff J.L. Heering H.A. Duin E.C. Albracht S.P. Armstrong F.A. Biochemistry. 1999; 38: 8992-8999Crossref PubMed Scopus (216) Google Scholar, 35Butt J.N. Filipiak M. Hagen W.R. Eur. J. Biochem. 1997; 245: 116-122Crossref PubMed Scopus (73) Google Scholar). Can PFOR serve as an efficient pyruvate synthase or is it prejudiced toward oxidative decarboxylation? One test of its catalytic bias is to compare the specificity factor (V/K) for the two opposing reactions. Another test is to determine the relative rates of the opposite reactions at physiological substrate concentrations. A significant catalytic preference in one direction would suggest that PFOR and pyruvate synthase might be separate enzymes. The difficulty in studying pyruvate synthesis from acetyl-CoA and CO2 is that a sufficiently strong electron donor must be coupled to drive this energetically demanding reaction, with a reduction potential below −540 mV. In the work reported here, we have coupled pyruvate synthesis to CO oxidation by CO dehydrogenase (Reaction 2 and Scheme FS1), which has a similarly low reduction potential (36Lindahl P.A. Münck E. Ragsdale S.W. J. Biol. Chem. 1990; 265: 3873-3879Abstract Full Text PDF PubMed Google Scholar). If PFOR and pyruvate synthase are the same enzyme, one conceivable way to regulate the metabolic direction of pyruvate metabolism is for PFOR/pyruvate synthase to use a high potential electron acceptor in the oxidative direction and a separate low potential donor in the reductive direction. For example, in the fumarate reductase and succinate dehydrogenase systems, the low potential donor menaquinol is a better substrate for fumarate reduction; whereas, ubiquinone, which has a higher midpoint potential, preferentially couples to succinate (32Maklashina E. Berthold D.A. Cecchini G. J. Bacteriol. 1998; 180: 5989-5996Crossref PubMed Google Scholar). We do not yet know which electron acceptor(s) are most efficient in the PFOR and pyruvate synthase reactions. Because rubredoxin rapidly accepts electrons from PFOR, it was proposed to be the physiological electron acceptor for the oxidative decarboxylation reaction, whereas reduced ferredoxin was designated as the electron donor for the synthase reaction (37Yoon K.S. Hille R. Hemann C. Tabita F.R. J. Biol. Chem. 1999; 274: 29772-29778Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, the kinetic parameters for the substrates and electron donors/acceptors in each direction were not rigorously studied leaving some questions about electron carrier specificity unanswered. Furthermore, the electron carrier specificity may be different in different organisms. C. thermoaceticum is an interesting model system for studying PFOR because the oxidative decarboxylation and reductive carboxylation reactions are essential for heterotrophic and autotrophic growth, respectively, of this organism. We recently characterized and defined the rate constants for the elementary steps in the C. thermoaceticum PFOR reaction (2Menon S. Ragsdale S.W. Biochemistry. 1997; 36: 8484-8494Crossref PubMed Scopus (62) Google Scholar). At high concentrations of substrates (k cat conditions), the synthase reaction was predicted to be ∼10-fold faster than the reverse reaction; however, the synthase reaction was not studied directly. Furthermore, the intracellular concentrations of the substrates and products of the PFOR reaction in C. thermoaceticum have not been determined; thus, the relative rates of the opposing reactions at physiological concentrations of substrates are unknown. Whether PFOR is an efficient pyruvate synthase for autotrophic acetogens thus remains an unanswered question. In the work reported here, we focused on several questions. What are the values of k cat andk cat/K m for the reverse reaction? Are these values consistent with a physiological function for PFOR in pyruvate synthesis? As a low potential electron donor, CO oxidation could conceivably provide sufficient strength to drive pyruvate synthesis. How effectively does the C. thermoaceticum pyruvate synthase reaction couple to the CO oxidation reaction? What is the most efficient direct electron donor and acceptor for the forward and reverse reactions of PFOR? N2 (99.8%, from Linweld) was deoxygenated by passage through a heated column containing BASF catalyst. CO (99.99%) was obtained from Linweld. Sodium bicarbonate (minimum 99.5%), acetyl-CoA (96%, sodium salt), β-nicotinamide adenine dinucleotide, reduced form (98%, disodium salt), andl-lactic dehydrogenase (type II: from rabbit muscle) were purchased from Sigma. C. thermoaceticum (Moorela thermoacetica) strain ATCC 39073 was grown anaerobically at 55 °C in a 14-liter fermentor on glucose and CO2. CODH, PFOR, and ferredoxin were purified under strictly anaerobic conditions. PFOR was purified as described (20Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google Scholar) in anaerobic 50 mm MOPS buffer at pH 7.5. CODH/ACS (38Ragsdale S.W. Clark J.E. Ljungdahl L.G. Lundie L.L. Drake H.L. J. Biol. Chem. 1983; 258: 2364-2369Abstract Full Text PDF PubMed Google Scholar) and ferredoxin (39Elliott J.I. Ljungdahl L.G. J. Bacteriol. 1982; 151: 328-333Crossref PubMed Google Scholar) were purified under anaerobic conditions at 17 °C in a vacuum atmospheres chamber maintained below 10 ppm of oxygen. Enzyme concentrations were determined by the Rose Bengal method (40Elliott J.I. Brewer J.M. Arch. Biochem. Biophys. 1978; 190: 351-357Crossref PubMed Scopus (90) Google Scholar) using a bovine serum albumin standard. CODH/ACS activity was assayed by monitoring the CO-dependent reduction of methyl viologen (ε604 = 13,900m−1 cm−1) (38Ragsdale S.W. Clark J.E. Ljungdahl L.G. Lundie L.L. Drake H.L. J. Biol. Chem. 1983; 258: 2364-2369Abstract Full Text PDF PubMed Google Scholar). PFOR activity was measured as described (2Menon S. Ragsdale S.W. Biochemistry. 1997; 36: 8484-8494Crossref PubMed Scopus (62) Google Scholar) by following pyruvate- and CoA-dependent methyl viologen reduction. Ferredoxin activity was measured by coupling CO oxidation by CODH/ACS to metronidazole reduction (ε320 = 9300m−1 cm−1) (42Terlesky K.C. Ferry J.G. J. Biol. Chem. 1988; 263: 4080-4082Abstract Full Text PDF PubMed Google Scholar) in a reaction similar to that described earlier for hydrogenase (43Chen J.S. Blanchard D.K. Anal. Biochem. 1979; 93: 216-222Crossref PubMed Scopus (58) Google Scholar). Rubredoxin was purified from Clostridium formicoaceticum (44Ragsdale S.W. Ljungdahl L.G. J. Bacteriol. 1984; 157: 1-6Crossref PubMed Google Scholar). Pyruvate synthase activity was determined in a coupled reaction with CO and CO dehydrogenase as the initial electron donor. Pyruvate formation was coupled to NADH oxidation by lactate dehydrogenase (LDH). Stock solutions of 1 mKHCO3, 10 mm or 1 mm acetyl-CoA, 22.4 mm NADH, 0.1 mm CODH, 33 μmPFOR, 121 μm ferredoxin, 10 mm methyl viologen, and 22 μm LDH were used. The assay was performed under an atmosphere of CO (1 mm) in a reaction mixture containing 1 μm CODH, 1 μmferredoxin or 40 μm methyl viologen, 110 nmLDH, 16.5 nm PFOR and varying concentrations of acetyl-CoA and CO2. The CO2 concentration was estimated by the Henderson-Hasselbach equation taking into account the pH of the reaction mixture and the KHCO3 concentration. During the initial velocity time-scale CO2 produced from CO was too low to affect the CO2 concentrations in solution. PFOR, CODH, electron carrier (ferredoxin, methyl viologen, or rubredoxin), and LDH concentrations were varied in a coupled reaction to establish the conditions under which pyruvate synthase activity is rate-limiting. In all assays, the buffer was 50 mm MES, pH 6.4, and saturated with CO gas by bubbling for 5 min with CO in a serum-stoppered cuvette. NADH oxidation (ε340 = 6400m−1 cm−1) was followed in an OLIS-modified Cary 14 spectrophotometer. The assays were performed at 25 °C and were initiated by adding PFOR. TheV max and K m values were determined by globally fitting the data to the equation for a ping-pong mechanism. PFOR has been shown to follow a ping-pong mechanism (21Uyeda K. Rabinowitz J.C. J. Biol. Chem. 1971; 246: 3120-3125Abstract Full Text PDF PubMed Google Scholar). The data were analyzed using Sigma Plot 5.0 (Jandel Scientific, San Rafael, CA). To determine the Michaelis parameters of PFOR for ferredoxin, the reaction mixture contained 10 mm pyruvate, 1 mm CoA, 1 mm thiamin pyrophosphate, 2 mm MgCl2, 0.1 mmmetronidazole, and varying concentrations of ferredoxin in 50 mm Tris-HCl buffer, pH 7.1. The reaction was started by adding 2 μl of PFOR (1.3 mg/ml) to a reaction volume of 0.5 ml, and the reduction of metronidazole was followed at 320 nm (ε320 = 9300 m−1cm−1). To determine the kinetic parameters for the reaction with rubredoxin, the reaction was started by adding 1 μl of PFOR (0.42 mg/ml) to a 0.2-ml reaction mixture, and the reduction of rubredoxin was followed at 490 nm (ε490 = 7300m−1 cm−1) (44Ragsdale S.W. Ljungdahl L.G. J. Bacteriol. 1984; 157: 1-6Crossref PubMed Google Scholar). The concentrations of reagents were the same as for the reaction with ferredoxin except that metronidazole was omitted. To determine the steady-state Michaelis parameters for pyruvate, the reaction mixture contained 1 mm CoA, 8 mm methyl viologen, 1 mm thiamin pyrophosphate, 2 mmMgCl2, in 50 mm Tris buffer, pH 7.4. The concentration of pyruvate was varied from 0.1 to 10 mm. The reduction of methyl viologen was followed at 604 nm. The data were fit to the Michaelis-Menten equation to yield the following parameters:V max = 14.2 units/mg,K m for pyruvate = 0.30 mm, andk cat/K m = 9.33 × 104m−1 s−1. C. thermoaceticum cells (2 g, 0.57 g of dry weight) in 5 ml of sonication buffer were sonicated for 5 min (15 s on, 45 s off). The sonication buffer contained 0.002 ng/ml DNase, 0.017 mg/ml phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme in 50 mm Tris-HCl, pH 7.6. After sonication, the proteins were precipitated with 2 m perchloric acid at 4 °C. After centrifugation at 14,000 rpm for 10 min, the pH was increased to pH 5.5 with 1 m NaOH, the solution was again centrifuged, and the concentrations of metabolites were measured in the supernatant. Radioactive acetyl-CoA (1 μl of a 850 μm stock solution with 96,950 dpm/nmol) was added to the supernatant as an internal control to determine recovery from the extraction procedure. More than 99% of the acetyl-CoA added was recovered after the final extraction step. The amounts of CoASH and acetyl-CoA were determined by reversed-phase high performance liquid chromatography on a Waters 616/626 LC System equipped with a Waters 996 PDA detector (Waters Inc.). We obtained the best separation of the cell extract components by eluting with a gradient from 5 to 50% methanol in 100 mm phosphate buffer, pH 5, over a 30-min time period. We used a C-18 μBondapack chromatographic column. Under these conditions, the retention times for CoASH and for acetyl-CoA were 10.3 and 13.3 min, respectively. The concentrations of CoASH and acetyl-CoA in the cell extract were estimated based on a standard curve (nmoles CoASH, acetyl-CoA versus peak area). The concentration of pyruvate was determined by the lactate dehydrogenase (45LeJohn H. Stevenson R. Methods Enzymol. 1975; 41: 293-298Crossref PubMed Scopus (7) Google Scholar) assay. An internal volume of 1 μl/mg protein (49Grupe H. Physiologische Ereignisse in Clostridium acetobutylicum beim Übergang von der Säure-zur Lösungsmittelbildung Ph.D. thesis. University of Goettingen, Goettingen, Germany1991Google Scholar) and a ratio of 0.6 for the mg of total protein/mg of dry weight (50Bahl H. Kontinuierliche Aceton-Butanol-Gärung durch Clostridium acetobutylicum Ph.D. thesis. University of Goettingen, Goettingen, Germany1983Google Scholar) were used in calculations of intracellular concentrations of metabolites. So for 0.57 g of dry weight of cells, we calculated 0.342 ml of total internal volume. Pyruvate Synthesis with PFOR—For convenience, we will refer to the pyruvate synthase or PFOR reactions explicitly but generally refer to the enzyme as PFOR. Pyruvate synthesis from acetyl-CoA and CO2 requires a strong electron donor. Because CO2 produced by the pyruvate decarboxylation reaction is the source of the carbonyl group in acetyl-CoA synthesis by clostridial CODH/ACS (20Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google Scholar), we coupled pyruvate synthesis to CO oxidation by CODH/ACS. The rate of pyruvate formation was monitored by the lactate dehydrogenase-coupled oxidation of NADH to NAD (SchemeFS1). It was earlier shown that the addition of an electron carrier stimulates the ability of PFOR to couple to CODH, giving a 5-fold increase in the rate of CO formation (20Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google Scholar). However, in the pyruvate synthase reaction, either ferredoxin or methyl viologen was required to mediate the electron transfer from reduced CODH/ACS to pyruvate synthase (Fig. 2). Given the complexity of this coupled system, it was essential to establish conditions under which pyruvate synthase is rate-limiting. These conditions include 1 μm CODH, 1 μm ferredoxin, or 40 μm methyl viologen, 110 nm LDH, 1 mm CO. At concentrations of PFOR at or below 16.5 nm, the reaction rate was strictly dependent upon PFOR. Different concentrations of CO2 (0.56–22.4 mm) and acetyl-CoA (0.005–0.05 mm) were used to define the kinetic parameters for the pyruvate synthase reaction (Fig.1).Figure 1Steady-state kinetics of the pyruvate synthase reaction. The assays were performed at 25 °C and were initiated by adding PFOR. The concentration of CO2 was varied at 5 μm (●), 10 μm (○), and 50 μm (▾) fixed concentrations of acetyl-CoA. The Michaelis parameters V max andK m were determined by global fitting of the data to a ping-pong mechanism. The Michaelis parameters determined wereV max 1.6 ± 0.1 unit/mg, KmAcetyl-CoA = 9.1 ± 1.4 μm, and KmCO2 = 2.0 ± 0.3 mm.View Large Image Figure ViewerDownload (PPT) The kinetic data did not fit a ternary complex mechanism; the kinetic parameters K ia and KmAcetyl-CoA had unreasonably large standard deviations (over 200% error). On the other hand, the data fit a ping-pong mechanism quite well (<15% standard deviations). The kinetic parameters were determined by globally fitting the data to a ping-pong mechanism. The Michaelis parameters for the pyruvate synthase are given in Table I along with those for the PFOR reaction, which we also determined. With CO and CODH as the electron donor, the pyruvate synthase activity is high. At saturating CO2 (22.4 mm) and acetyl-CoA (100 μm) concentrations, the specific activity is 1.6 unit/mg, which translates to a k cat of 3.2 s−1. This is only 9-fold lower than thek cat for the PFOR reaction (28 s−1), which agrees well with predictions based on kinetic simulations (2Menon S. Ragsdale S.W. Biochemistry. 1997; 36: 8484-8494Crossref PubMed Scopus (62) Google Scholar). When methyl viologen was replaced by ferredoxin (1 μm) at saturating concentrations of pyruvate and CoA, there was no difference in the specific activity, indicating that the second half reaction of pyruvate synthesis, not electron donation to the enzyme, is rate-limiting under these conditions.Table IKinetic parameters for the pyruvate synthase and PFOR reactionsSubstrateK mk catk cat/K mExpected rate1-aCalculated rate at physiological concentrations of substrates.Measured ratedRef.mms −1mm −1 s −1s −1s −1Acetyl-CoA1-bPyruvate synthase reaction.9 × 10−33.23563.21.3This workCO21-bPyruvate synthase reaction.2.03.21.63.2This workCoASH1-cRefers to the PFOR reaction.4 × 10−3287 × 1032810.820Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google ScholarPyruvate1-cRefers to the PFOR reaction.0.302893.318.6This workFerredoxin1-cRefers to the PFOR reaction.3.2 × 10−429.69.24 × 104This workFerredoxin1-bPyruvate synthase reaction.2.7 × 10−42.07.4 × 103This workRubredoxin1-cRefers to the PFOR reaction.9.4 × 10−319.82.1 × 103This workRubredoxin1-bPyruvate synthase reaction.<0.1This workMethyl viologen1-cRefers to the PFOR reaction.1.118.71720Menon S. Ragsdale S.W. Biochemistry. 1996; 35: 12119-12125Crossref PubMed Scopus (43) Google Scholard Measured rates at intracellular concentrations of substrates.1-a Calculated rate at physiological concentrations of substrates.1-b Pyruvate synthase reaction.1-c Refers to the PFOR reaction. Open table in a new tab d Measured rates at intracellular concentrations of substrates. Initial velocity experiments were used to determine the physiological electron carriers(s) for PFOR (Fig.2 A) and pyruvate synthase (Fig. 2 B). With ferredoxin as the mediator, thek cat/K m for ferredoxin for the synthase reaction is only ∼10-fold lower than for the oxidative decarboxylation. The 8-iron ferredoxin from the archaeon,Methanosarcina thermophila, also was as good an electron mediator as the C. thermoaceticum ferredoxin (Fig.3), suggesting that these proteins are homologous enough to interact in a similar way with both PFOR and CODH/ACS. Recently, it was proposed for the PFOR of C. tepidum that rubredoxin is the electron acceptor, whereas ferredoxin is the electron donor for the pyruvate synthase reaction (37Yoon K.S. Hille R. Hemann C. Tabita F.R. J. Biol. Chem. 1999; 274: 29772-29778Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This hypothesis is reasonable because rubredoxin has a midpoint potential near 0 mV (inC. tepidum it is −87 mV); whereas, ferredoxins have much more negative midpoint potentials, generally below −400 mV (46Stephens P.J. Jollie D.R. Warshel A. Chem. Rev. 1996; 96: 2491-2513Crossref PubMed Scopus (295) Google Scholar). However, the values of V/K, the so-called specificity factors, for rubredoxin and ferredoxin in the pyruvate synthase or PFOR reactions have never been compared. The value ofk cat/K m for rubredoxin in the oxidative decarboxylation reaction is 2.1 × 106m−1 s−1 (Fig.4), which is 50-fold lower than that for ferredoxin. Thus, although the driving force for reducing rubredoxin with pyruvate is much stronger in C. thermoaceticum, there is a strong preference of PFOR for ferredoxin in the oxidative decarboxylation reaction. Rubredoxin could not couple CO oxidation to the pyruvate synthase reaction (Table I and Fig. 3). Because rubredoxin is a highly active electron acceptor for CODH (38Ragsdale S.W. Clark J.E. Ljungdahl L.G. Lundie L.L. Drake H.L. J. Biol. Chem. 1983; 258: 2364-2369Abstract Full Text PDF PubMed Google Scholar), the bottleneck is in the pyruvate synthase step. This is reasonable because the standard reduction potential for the Fe3+/Fe2+ couple of rubredoxin is much more positive than that of the acetyl-CoA + CO2/pyruvate couple. To assess the physiological relevance of the pyruvate synthase reaction of PFOR, we determined the pyruvate, acetyl-CoA, and CoASH concentrations in growing C. thermoaceticum cells (Table I). The amounts of these metabolites in the cell extract, determined by HPLC, were related to the intracellular volume (0.57 g of cell dry weight = 0.342 ml of cell internal volume) (TableII). We estimate the intracellular concentrations of CoASH and acetyl-CoA to be 0.28 and 0.01 mm, respectively. The intracellular pyruvate concentration is 0.2 mm, whereas the CO2 concentration is estimated to be 33 mm, because cells are continually sparged with 100% CO2 during growth.Table IIThe intracellular concentration of pyruvate, coenzyme A, and acetyl-coenzyme A in C. thermoaceticumMetabolitesIntracellular concentrationMethod usedμmPyruvate180 ± 40 (n = 3)Lactate dehydrogenase assayCoenzyme A280 ± 90 (n = 3)HPLCAcetyl-Coenzyme A10.4 ± 3.5 (n = 3)HPLC Open table in a new tab Because the intracellular pyruvate concentration is below its K m value (0.3 mm), the PFOR reaction would be limited by the concentration of pyruvate. Similarly, the pyruvate synthase reaction would be limited by the concentration of acetyl-CoA in the cell. The consequence is that, at physiological concentrations of pyruvate and acetyl-CoA, the carboxylation of acetyl-CoA is predicted to occur about 8-fold slower than oxidative decarboxylation of pyruvate. To verify these predicted values, we ran the forward and reverse reactions in the presence of the measured intracellular concentrations of pyruvate, coenzyme A, acetyl-CoA, and CO2 (Table I). The results confirmed the prediction with a ratio of oxidative decarboxylation to reductive carboxylation of 8.3. The oxidative decarboxylation of pyruvate by PFOR generates low potential electrons that can be coupled to important reactions in the C, N, S, and H cycles, e.g. dinitrogen reduction, proton reduction to H2, sulfate reduction, and CO2reduction. PFOR also generates the key metabolic intermediate, acetyl-CoA. Thus, this important reaction has been studied rather extensively. The reverse reaction, reduction of acetyl-CoA to pyruvate, is equally important for autotrophic anaerobes because it links the Wood-Ljungdahl pathway of acetyl-CoA formation to the incomplete reductive tricarboxylic acid cycle for synthesis of longer carbon biosynthetic precursors. Anabolic reactions in acetogens are not well studied; however, in methanogens, it is clear that the incomplete tricarboxylic acid cycle converts oxaloacetate (derived from pyruvate by the action of pyruvate carboxylase or the linked activities of phosphoenolpyruvate synthetase and phosphoenolpyruvate carboxylase) into malate, fumarate, succinate, succinyl-CoA, and α-ketoglutarate (10Simpson P.G. Whitman W.B. Ferry J.G. Methanogenesis: Ecology Physiology, Biochemistry & Genetics. Chapman & Hall, London1993: 445-472Crossref Google Scholar). What are the properties of the enzyme responsible for the carboxylation of acetyl-CoA? Evidence described in the Introduction is consistent with the concept that PFOR also serves as a pyruvate synthase in acetogenic and methanogenic microbes. The results described here support that hypothesis. The C. thermoaceticumPFOR is a very active pyruvate synthase, withk cat andk cat/K m values only 8–10-fold lower than those for PFOR. At physiological concentrations of pyruvate, CoA, acetyl-CoA, and CO2, the pyruvate synthase activity is about 8-fold slower than PFOR. This rate is sufficient for the rate of biosynthesis by growing cells, because the flux of metabolic intermediates toward biosynthesis is significantly lower than the flux toward energy generation. This may be even more marked for anaerobic organisms, which are energy limited. For example, only 4% of the CO2 consumed during the growth ofC. thermoaceticum on CO2 and H2as the sole energy and carbon source is recovered in cell biomass (47Daniel S.L. Hsu T. Dean S.I. Drake H.L. J. Bacteriol. 1990; 172: 4464-4471Crossref PubMed Scopus (143) Google Scholar). When cells were grown on glucose, 7% of the substrate consumed is recovered in cell biomass; most of the remainder is retrieved in acetate. In summary, it appears that the pyruvate synthase activity of PFOR is sufficient to account for its role in the generation of pyruvate as a biosynthetic precursor. What is the physiological electron carrier(s) for pyruvate synthase and PFOR? With a midpoint potential for the acetyl-CoA + CO2/pyruvate couple of −520 mV, pyruvate is a strong reductant. Correspondingly, it requires a very strong reductant to carboxylate acetyl-CoA. Tabita (37Yoon K.S. Hille R. Hemann C. Tabita F.R. J. Biol. Chem. 1999; 274: 29772-29778Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) discovered that rubredoxin is a strong electron acceptor for the PFOR from the green sulfur bacterium C. tepidum, indicating that it could be the physiological redox mediator in this direction. The C. thermoaceticum CODH is a highly promiscuous electron donor and can rapidly reduce rubredoxin. Several roles of rubredoxin in anaerobic energy metabolism have been proposed. These have been recently discussed in relation to a new proposed role for rubredoxin in protecting cells from oxygen (41Gomes C.M. Silva G. Oliveira S. LeGall J. Liu M.Y. Xavier A.V. Rodrigues-Pousada C. Teixeira M. J. Biol. Chem. 1997; 272: 22502-22508Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 48Jenney Jr., F.E. Verhagen M.F. Cui X. Adams M.W. Science. 1999; 286: 306-309Crossref PubMed Scopus (333) Google Scholar). Rubredoxin is expected to be a very good electron acceptor from pyruvate, because its Fe3+/2+ couple is near 0 mV. Therefore, on the basis of driving force alone, rubredoxin could accept electrons six orders of magnitude faster than ferredoxin (midpoint potential of about −350 to −400 mV). However, thek cat/K m for rubredoxin 3We used rubredoxin from C. formicoaceticum not C. thermoaceticum to couple to theC. thermoaceticum PFOR, because this protein is relatively abundant in iron-limited cultures of C. formicoaceticum. These small electron transfer mediators can generally be used fairly interchangeably among related microbes. We do not expect this to significantly alter our conclusions, because the C. formicoaceticum rubredoxin couples equally well to the C. formicoaceticum and C. thermoaceticum CODH. Furthermore, the M. thermophila ferredoxin is nearly as good an electron acceptor as the C. thermoaceticum protein. in pyruvate oxidation is 50-fold lower than that for ferredoxin. Thus, although the driving force for ferredoxin reduction is much less relative to rubredoxin, ferredoxin is a much more efficient acceptor of electrons from PFOR. An apparently more serious problem with rubredoxin as an electron acceptor for PFOR is that it is a very poor electron donor for subsequent reactions. Therefore, it seems unreasonable to expect rubredoxin to couple pyruvate oxidation to proton, CO2, or dinitrogen reduction. The CO2/CO and the CO2 + acetyl-CoA/pyruvate couples have similar standard reduction potentials. Accordingly, we found that rubredoxin is unable to couple CO oxidation to the pyruvate synthase reaction. On the other hand, ferredoxin is an excellent mediator in this reaction. Although, rubredoxin is inept as an electron donor for pyruvate synthesis, the value ofk cat/K m for ferredoxin in the synthase reaction is only ∼10-fold lower than in the oxidative decarboxylation. Thus, our results indicate that PFOR is an efficient pyruvate synthase and that ferredoxin serves as the electron acceptor for pyruvate oxidation as well as the electron donor for pyruvate synthesis." @default.
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• W2046287787 title "The Role of Pyruvate Ferredoxin Oxidoreductase in Pyruvate Synthesis during Autotrophic Growth by the Wood-Ljungdahl Pathway" @default.
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