In an effort to better understand nitrogenase—the essential biocatalyst that bacteria use to convert N2 into ammonia—researchers have now solved a mystery concerning the origins of a carbide ion in the enzyme complex. The work could lead to engineered versions of the enzyme that catalyze nitrogen fixation and related reactions for industrial applications such as ammonia production.
A decade ago, structural biologist Douglas C. Rees of Caltech and coworkers discovered a light atom of unknown identity in nitrogenase’s FeMo cofactor, the enzyme’s catalytic center. The atom seemed important because it was bonded to six of the cofactor’s seven iron atoms.
Last year, two groups showed independently that the mystery atom is carbide (C4–). Such carbide-iron species are extremely rare in biological metal centers. One group included X-ray spectroscopist Serena DeBeer of Cornell University and Max Planck Institute for Bioinorganic Chemistry, in Germany, and chemical biologist Markus W. Ribbe of the University of California, Irvine; and the other was led by metalloprotein expert Oliver Einsle of the University of Freiburg, in Germany.
Researchers have since been speculating about whether the nitrogenase-related protein NifB is responsible for inserting carbide into the FeMo cofactor. But finding answers has been difficult because NifB is too unstable to work with experimentally.
Now, Ribbe, colleague Yilin Hu, and coworkers at UC Irvine have nailed down the origins of the carbide. To do so, they linked NifB to another complex, NifEN. This linkage stabilized NifB, enabling them to show that NifB transfers carbide from S-adenosylmethionine to the FeMo cofactor during biosynthesis and to describe possible carbide-insertion mechanisms (Science, DOI: 10.1126/science.1224603).
“This fundamentally advances our understanding of a very complex process, as the FeMo cofactor is the most complicated biological metal cluster we know,” Einsle comments. “NifB was suspected to be involved in carbide transfer, but it was Hu, Ribbe, and coworkers who came up with the decisive experiments. I find it highly original that they managed to stabilize NifB by fusing it to NifEN. The experiments are brilliant—simple and yet unambiguous.” Further research in this area, he says, could put to rest remaining questions about how the FeMo cofactor is assembled. It could also provide answers to “the big questions of how and where on the FeMo cofactor the nitrogenase reaction actually takes place, ” he adds.
“I don’t see how anyone cannot feel that we have entered a new era in understanding nitrogenase, with the demonstration by chemical and biochemical methods of the origin of the enigmatic interstitial ligand,” Rees says. The work “should provide insights into new approaches for the chemical synthesis of cofactor analogs, including modified versions that could have altered substrate-reduction properties.”