This award in the Chemistry of Life Processes (CLP) program in the Division of Chemistry at NSF supports a collaborative project between Professor Alan A. DiSpirito at Iowa State University and Professor Eckard Munck at Carnegie Mellon University to carry out fundamental studies aimed at elucidating the structure of the active site and the reactivity of the so-called particulate form of methane monooxygenase (pMMO). This is a ubiquitous bacterial enzyme that mediates the conversion of methane to methanol and thus plays a key role in the global carbon cycle. The selective oxidation of methane to methanol using mild reaction conditions is one of the most important industrial processes pursued by organometallic chemists and could be a direct application of the structural elucidation of pMMO's active site. The project includes the extensive use of Mössbauer spectroscopy to characterize the purported diiron center in pMMO, the identification of catalytic and electron transfer centers using Electron Paramagnetic Resonance (EPR) spectroscopy, and a mechanistic investigation of methane oxidation. This is an eminently interdisciplinary project at the interface of biochemistry, microbial physiology, and biophysical chemistry and will expose undergraduate and graduate students to a variety of techniques, ranging from protein purification to EPR spectroscopy.
The broader impacts of the proposal are focused on the training of students, including members from underrepresented groups and undergraduate students from the University of Wisconsin-Eau Claire, a Predominantly Undergraduate Institution.
Methanotrophs are characterized by the capacity to utilize methane as a sole carbon and energy source. This bacterial group have received increasing attention given that methane is a very potent greenhouse gas, with a global warming potential 28-34 times that of carbon dioxide over a 100-year period, and methanotrophs are estimated to remove up to 90% of the methane produced in anaerobic soils. The first step in methane oxidations involves the conversion of methane to methanol, which is catalyzed by methane monooxygenase of which there are two known forms. One, the membrane-associated, or particulate methane monooxygenase, is a found in most known methanotrophs and is located in the cytoplasmic membrane or particulate cell fractions. The other is the soluble methane monooxygenase, which is found in the cytoplasm or soluble fractions of some, but not all methanotrophs. The two monooxygenases are genetically and structurally distinct enzymes. In methanotrophs that have both forms of methane monooxygenase, copper is known to regulate the expression of the genes encoding these methane monooxygenases as well as their enzymatic activities. A diiron center has been shown to be the catalytic site of the soluble methane monooxygenase. All laboratories studying the membrane associated methane monooxygenase agree that it is a copper-containing enzyme. However, there is a disagreement among researchers as to the number, type, and function of the metal centers associated with the membrane associated methane monooxygenase, as well as to the nature of the physiological electron donor in the reaction. However, higher activity preparations of the membrane associated methane monooxygenase must be developed before this question can be addressed. Previous results have demonstrated that high copper concentrations during cell growth stabilizes the membrane associated methane monooxygenase and that iron and copper uptake in M. capsulatus Bath are linked. Results from the past two years of this proposal have shown that copper uptake is also influenced by iron uptake. In an attempt to increase the stability of the membrane associated methane monooxygenase in cell free fractions the concentrations of copper and iron during growth were manipulated to maximize activity and minimize adventitiously bound metals. To meet these aims we reduced the final concentration of both iron and copper in the culture media by 50%. Expression of the membrane-associated methane monooxygenase under these culture conditions was reduced by approximately 20%. However, the percent recovery of membrane-associated methane monooxygenase activity in the cell free fraction was over twice the recovery of cells cultured in media containing higher copper concentration. Using this modified culture procedure as well as cell lysis condition, we now recover 70 – 80% of the whole cell membrane-associated methane monooxygenase activity in the cell free fraction. We are now in a good position to determine the metal composition and catalytic site of this enzyme. Previous studies suggested methanobactin may be the respiratory link between the membrane-associated methane monooxygenase and the respiratory chain. Methanobactins are low molecular mass (≈ 1 kDa), copper-ligating molecules produced by many, but not all methanotrophs, and released into the surroundings to scavenge for copper. To approach this question we deemed it necessary to generate a methanobactin minus mutant. However, before we could generate methanobactin minus mutant we needed to determine how this small highly modified protein was synthesized. Initially we believed the molecule was synthesized via a non-ribosomal peptide synthetase (NRPS) or via a polyketide synthase (PKS). However, mutations in the two known NRPS operons and the one PKS operon identified in M. capsulatus Bath genome did not disrupt the production of methanobactin. The clue to identifying the structural gene for methanobactin can following hydrolysis of the two oxazolone rings in the methanobactin from M. trichosporium OB3b. The 11 amino acid peptide produced following the hydrolysis lead to the identification of a gene of unknown function. Generation of a mutation in this gene produced a methanobactin minus mutant and resulted in: (1) the identified the structural gene for methanobactin, (2) the putative biosynthetic operon for methanobactin, (3) identification of methanobactin-like structural genes and biosynthetic operons in a number of methanotrophs and non-methanotrophs and (4) a testable model on how the two methane monooxygenases are regulated in methanotrophs. The mutant also demonstrated that methanobactin stimulates methane oxidation by the membrane-associated methane monooxygenase but is not essential for activity.
