Collaborative Research: Mechanistic Insights into Substrate Transformations by the Complex Iron-Sulfur Molybdoenzyme (CISM) Family

Researchers at Indiana University Indianapolis and the University of Wisconsin–Milwaukee are investigating a family of metal-containing enzymes called complex iron-sulfur molybdoenzymes (CISM), which play key roles in the natural cycling of elements like carbon, nitrogen, sulfur, and arsenic. These enzymes enable many microorganisms to obtain energy and survive under changing environmental conditions, making them important for both ecosystem function and global biogeochemical cycles. The research focuses on the enzymes' catalytic center, known as the molybdenum cofactor, to understand how these enzymes recognize specific molecules, control chemical reactions, and determine whether reactions proceed in the forward or reverse direction. By combining biochemical, spectroscopic, structural, and computational approaches, the team will examine how amino acids surrounding the active site influence enzyme function. The project will advance fundamental knowledge of how molybdenum enzymes work and provide a foundation for future efforts to harness these catalysts for environmental and industrial applications, including carbon dioxide conversion, water treatment, and more sustainable nitrogen-based chemical production. The project will also provide interdisciplinary research training and professional development opportunities for undergraduate, graduate, and postdoctoral researchers, helping prepare the next generation of scientists in chemistry and the biological sciences. The complex iron-sulfur molybdoenzyme (CISM) family of enzymes is the largest group of enzymes that use the molybdenum cofactor (Moco) at the catalytic center. Although several reaction mechanisms have been proposed, experimental evidence supporting these models is limited, and many individual reaction steps remain uncertain. In many cases, the individual steps are debated. Some CISM enzymes can even work in both directions, i.e., reversibly. A long-standing idea is that small changes in the enzyme’s structure control which substrates it prefers and whether its reactions are reversible. This project will study a representative enzyme, periplasmic nitrate reductase (NapA). NapA converts nitrate into nitrite, a critical and slow step in processes like denitrification and ammonia production. A multidisciplinary approach spanning enzyme kinetics, structural studies, spectroscopy, and computation will provide a multifaceted description of the Moco and the reactions catalyzed. This work aims to provide a clearer, fundamental understanding of how Moco-containing enzymes prefer their substrates and control reaction pathways and direction. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.