Metal centers in the anaerobic microbial metabolism of CO and CO2.

Carbon dioxide and carbon monoxide are important components of the carbon cycle. Major research efforts are underway to develop better technologies to utilize the abundant greenhouse gas, CO(2), for harnessing 'green' energy and producing biofuels. One strategy is to convert CO(2) into CO, which has been valued for many years as a synthetic feedstock for major industrial processes. Living organisms are masters of CO(2) and CO chemistry and, here, we review the elegant ways that metalloenzymes catalyze reactions involving these simple compounds. After describing the chemical and physical properties of CO and CO(2), we shift focus to the enzymes and the metal clusters in their active sites that catalyze transformations of these two molecules. We cover how the metal centers on CO dehydrogenase catalyze the interconversion of CO and CO(2) and how pyruvate oxidoreductase, which contains thiamin pyrophosphate and multiple Fe(4)S(4) clusters, catalyzes the addition and elimination of CO(2) during intermediary metabolism. We also describe how the nickel center at the active site of acetyl-CoA synthase utilizes CO to generate the central metabolite, acetyl-CoA, as part of the Wood-Ljungdahl pathway, and how CO is channelled from the CO dehydrogenase to the acetyl-CoA synthase active site. We cover how the corrinoid iron-sulfur protein interacts with acetyl-CoA synthase. This protein uses vitamin B(12) and a Fe(4)S(4) cluster to catalyze a key methyltransferase reaction involving an organometallic methyl-Co(3+) intermediate. Studies of CO and CO(2) enzymology are of practical significance, and offer fundamental insights into important biochemical reactions involving metallocenters that act as nucleophiles to form organometallic intermediates and catalyze C-C and C-S bond formations.

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural consequences of the L8R mutation in 4-oxalocrotonate tautomerase.

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.