Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase.

A single‐well, “push‐pull” test method is proposed for the in situ determination of microbial metabolic activities in ground‐water aquifers. The method consists of the pulse‐type injection (“push”) of a test solution into the saturated zone of an aquifer through the screen of an existing monitoring well followed by the extraction (“pull”) of the test solution/ground‐water mixture from the same well. The test solution contains a tracer and one or more reactive solutes selected to investigate specific microbial activities. During the injection phase, the test solution flows radially away from the monitoring well into the aquifer. Within the aquifer, biologically reactive components of the test solution are converted to various products by the indigenous microbial community. During the extraction phase, flow is reversed and solute concentrations are measured to obtain breakthrough curves, which are used to compute the quantities of reactant(s) consumed and/or product(s) formed during the test and reaction rates. Tests were performed to determine rates of aerobic respiration, denitrification, sulfate reduction, and methanogenesis in a petroleum contaminated aquifer in western Oregon. High rates of oxygen, nitrate, nitrite, and hydrogen utilization and nitrite, and carbon dioxide production support the hypothesis that petroleum contamination has resulted in an increase in microbial activity in the anaerobic portion of the site. The results suggest that the push‐pull test method should be useful for obtaining quantitative information on a wide range of in situ microbial processes.

The initial reactions in the cometabolic oxidation of the gasoline oxygenate, methyl tert-butyl ether (MTBE), by Mycobacterium vaccae JOB5 have been characterized. Two products, tert-butyl formate (TBF) and tert-butyl alcohol (TBA), rapidly accumulated extracellularly when propane-grown cells were incubated with MTBE. Lower rates of TBF and TBA production from MTBE were also observed with cells grown on 1-or 2-propanol, while neither product was generated from MTBE by cells grown on casein-yeast extract-dextrose broth. Kinetic studies with propane-grown cells demonstrated that TBF is the dominant (>80%) initial product of MTBE oxidation and that TBA accumulates from further biotic and abiotic hydrolysis of TBF. Our results suggest that the biotic hydrolysis of TBF is catalyzed by a heat-stable esterase with activity toward several other tert-butyl esters. Propane-grown cells also oxidized TBA, but no further oxidation products were detected. Like the oxidation of MTBE, TBA oxidation was fully inhibited by acetylene, an inactivator of short-chain alkane monooxygenase in M. vaccae JOB5. Oxidation of both MTBE and TBA was also inhibited by propane (K i ‫؍‬ 3.3 to 4.4 M). Values for K s of 1.36 and 1.18 mM and for V max of 24.4 and 10.4 nmol min ؊1 mg of protein ؊1 were derived for MTBE and TBA, respectively. We conclude that the initial steps in the pathway of MTBE oxidation by M. vaccae JOB5 involve two reactions catalyzed by the same monooxygenase (MTBE and TBA oxidation) that are temporally separated by an esterase-catalyzed hydrolysis of TBF to TBA. These results that suggest the initial reactions in MTBE oxidation by M. vaccae JOB5 are the same as those that we have previously characterized in gaseous alkane-utilizing fungi.Methyl tert-butyl ether (MTBE) is presently an important component of many gasoline formulations used in the United States and several other countries. Low concentrations of MTBE (Ͻ3% vol/vol) were first added to gasoline in the United States in the 1980s to enhance the octane rating of unleaded gasoline. More recently, MTBE has been added as one of several oxygenating compounds intended to reduce automobile emissions of carbon monoxide and smog-related air pollutants. For this purpose MTBE is added to gasoline at concentrations in excess of 10% (vol/vol). The widespread use of MTBE and its recent detection in many urban groundwater supplies (44) have prompted concerns over the human health effects of chronic exposure to this compound through contaminated water supplies (26). The U.S. Environmental Protection Agency presently classifies MTBE as a possible human carcinogen and has issued a drinking water advisory for MTBE of 20 to 40 g/liter (parts per billion) (48).Ether bonds (49) and branched hydrocarbon skeletons (3) are features found in many compounds that are persistent in the environment. Although initial studies suggested that MTBE was not subject to extensive biodegradation under anaerobic conditions (35,53), it is now known to be degraded, albeit slowly, under methanogenic (51), s...

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.

effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. eluropaea. MATERIALS AND METHODS Growth and preparation of cells. Cells of N. europaea ATCC 19718 were grown in batch cultures (1 to 2 liters) and harvested by centrifugation as described previously (15).