The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.

30. Spectrophotometry indicated that Ph,PO had no effect on the reaction. The results thus far require that MoO(sap)(DMF) quantitatively reduce Mo02(L-NS2). This point was spectrophotometrically established with use of an equimolar D M F reaction system at ambient temperature.The foregoing results are summarized in Figure 8, which depicts spontaneous intermetal oxo-transfer reactions. In the set of complexes, MoO(sap)(DMF) (17) is the strongest reductant and M o O~( S~C N E~~)~ (19) is the strongest oxidant. This thermodynamic series is the same as kinetic series 19, thereby showing that the activation barrier to oxo transfer is largely set by those factors which stabilize/destabilize Mo(IV) and Mo(V1). This in turn reemphasizes the beneficial effect of anionic sulfur ligands in stabilizing Mo(IV). Lastly, the reactions 17 + ]/,02 -13 and 18 + 1/202 -15 cannot be placed precisely in the oxidative enthalpy series of Table 111. However, it is clear that the first of these reactions lies below the second and that the AH values of both are more negative than that for the oxidation of MoO-(L-NS2)(DMF). The lack of reaction between 17 and 10 equiv of Ph3P0 in DMF for 6 h at ambient temperature suggests that AH k -67 kcal/mol, but slow reaction kinetics cannot be ruled out. In any case, 17 and 18, as MoO(L-NS2)(DMF) and MoO-(S,CNEt2),, should reduce MezSO to Me#. This has been confirmed for the stronger reductant 17, which is quantitatively oxidized to 13 in a system initially containing 2 equiv of Me2S0. All MoIVO complexes in Figure 8 are now recognized to be thermodynamically competent to reduce Me2S0,75 the most re-ductively resistant enzyme substrate for which thermodynamic data are available. The stoichiometric reduction of substrate XO by a MoIVO complex is, therefore, a highly necessary but not a sufficient thermodynamic criterion for a functional oxo-transferase site model. What is required for sufficiency under the oxo atom transfer hypothesis are those factors which permit at least one such atom transfer to or from substrate followed by regeneration of the original Mo'"0 or MoV'02 species either by electron or oxo transfer, such that catalysis is sustained. The results presented here show that anionic sulfur ligation is a critical modulator of these factors and, as already mentioned, appears to place real or effective Mo redox potentials in a range accessible to physiological reactants.Ongoing research on biologically related oxo-transfer reactions includes development of catalytic systems for substrate oxidation and reduction, examination of reactions in aqueous solution, and the possible role of pterins in enzymic electron transfer. Acknowledgment. (75) In the only other related case that has been reported, [MoO(dttd)CIJ1reduces Me2SO: Kaul, B. B.; Enemark, J. H.; Merbs, S. L.; Spence, J. T. J . Am. Chem. SOC. 1985, 107, 2885. dttd = 2,3:8,9-dibenzo-1,4,7,lO-tetrathiadecane( 2-). Abstract: The syntheses, properties, and spectral characterization of the first examples of molecular hydrogen complexes, M(CO),(...

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The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.