PubMed · 7264631
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H M Jagannatha, P S Sastry. 1981. Cholesterol-esterifying enzymes in developing rat brain.. https://doi.org/10.1111/j.1471-4159.1981.tb00572.x
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Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV-visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., & Lindahl, P. A. (1996) Biochemistry 35, 8371-8380] that the Cred2 form of cluster C is two electrons more reduced than the Cred1 form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [Cred1,Box, Xox] state to yield a Cred1-CO complex; (ii) two-electron reduction of Cred1 to Cred2 concerted with CO2 release; (iii) binding of a second CO molecule to the [Cred2,Box,Xox] state to form a Cred2-CO complex; (iv) electron transfer from Cred2-CO to cluster B to form [Cred2,Bred,Xred] with concerted release of the second CO2. Step iii competes with internal electron transfer from Cred2 to Box and Xox. At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. Xox is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The Cred2 state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.
Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.
In the final reaction of peroxisomal alpha-oxidation of 3-methyl-branched fatty acids a 2-hydroxy-3-methylacyl-CoA intermediate is cleaved to formyl-CoA and a hitherto unidentified product. The release of formyl-CoA suggests that the unidentified product may be a fatty aldehyde. When purified rat liver peroxisomes were incubated with 2-hydroxy-3-methylhexadecanoyl-CoA 2-methylpentadecanal was indeed formed. The production rates of formyl-CoA (measured as formate) and of the aldehyde were in the same range. While the production of formate remained unaltered in the presence of NAD+, the amount of 2-methylpentadecanal was decreased, which was accompanied by the formation of 2-methylpentadecanoic acid. These data indicate that (1) during alpha-oxidation the 2-hydroxy-3-methylacyl-CoA is cleaved to a 2-methyl-branched aldehyde and formyl-CoA and (2) liver peroxisomes are capable of converting this aldehyde to a 2-methyl-branched fatty acid.