Metal Cofactor Composition and Coordination in the Hydrocarbon Synthesizing Methylthio-alkane Reductase
EMSL Project ID
60643
Abstract
Biological hydrocarbon production is essential for realizing a renewable bioeconomy of platform chemicals, such as methane and ethylene. For example, currently nearly all ethylene for the manufacturing of plastics, textiles, and detergents is produced from fossil fuels by energy intensive processes representing a significant portion of industrial global greenhouse emissions. Recently we discovered a reductase present in numerous proteobacteria and firmicutes that converts ubiquitous volatile organic sulfur compounds into hydrocarbons like methane, ethane, and ethylene. This enzyme complex called “methylthio-alkane reductase” is from the nitrogenase-like family of enzymes. As with true nitrogenases, methylthio-alkane reductase employs a homolog of the nitrogenase cofactor synthesis protein (NifB), nitrogenase reductase iron protein (NifH), and nitrogenase catalytic subunits (NifD and NifK) for assembly and activity. Initial purification, structural determinations, and catalytic analysis indicates that like true nitrogenases, the methylthio-alkane reductase catalytic subunits (MarD and MarK) employ sophisticated iron-sulfur clusters akin to the P-cluster and M-cluster of NifD and NifK. This distinguishes methylthio-alkane reductase from other known nitrogenase-like systems (bacteriochlorophyll and F430 cofactor reductases) which only employ 4Fe-4S clusters. Currently, the precise identity of the methylthio-alkane reductase metallocofactors and the amino acid residues that coordinate them are unknown. In this project we will employ inductively coupled plasma mass spectroscopy (ICP-MS) of purified methylthio-alkane reductase MarD and MarK complex to quantify the identity and ratio of metallocofactor elements, particularly iron, sulfur, and if present molybdenum or vanadium. Three different isoforms of nitrogenase metallocofactors are distinguished by a single molybdenum, vanadium, or iron substituent, and exhibit differing ATP hydrolysis and catalytic efficiencies. Identification of molybdenum, vanadium, or only iron in the methylthio-alkane reductase catalytic complex will provide needed insight into its structure and catalytic underpinnings. In addition, we will perform native electrospray mass spectroscopy (native MS) of the purified methylthio-alkane reductase. The resulting primary mass masses will help enumerate which protein subunits are involved in binding the metallocofactors. This will provide key information on the location and coordination of the metallocofactors, which in turn will help define the structure and function of methylthio-alkane reductase. Such information is critical for understanding the mechanism by which methylthio-alkane reductase catalyzes the synthesis of renewable ethylene. Ultimately, understanding of the metallocofactor identity and coordination will enable future engineering endeavors to increase the catalytic activity of this system for maximal methane and ethylene yields and to expand the product profile of this system.
Project Details
Project type
Limited Scope
Start Date
2023-01-18
End Date
2023-10-27
Status
Closed
Released Data Link
Team
Principal Investigator