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The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the [alpha]-subunit of butane monooxygenase hydroxylase (BMOH-[alpha]) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes ICEs) were reduced in mutant strain Gll3N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain Gll3N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G 113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain GI13N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent [O.sub.2] uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-[alpha] mutants might allow insights into the catalytic mechanism of BMO to be obtained.

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase [alpha] subunit of butane monooxygenase (BMOH-[alpha]) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-[alpha] were altered to the corresponding residues from the o~ subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains GI13N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G 113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to l-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 [micro]M methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 [micro]M. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-[alpha]