Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy

• Published in: The Journal of biological chemistry Vol. 288; no. 22; pp. 15532 - 15536
• Main Authors: Gumataotao, Natalie, Kuhn, Misty L., Hajnas, Natalia, Holz, Richard C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-05-2013; American Society for Biochemistry and Molecular Biology
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Catalysis; Catalytic Domain; Enzyme Catalysis; Enzyme Mechanisms; Enzymology; Hydro-Lyases - chemistry; Hydro-Lyases - metabolism; Iron; Kinetics; Models, Chemical; Nitrile hydratase; Rhodococcus equi - enzymology; Spectroscopy; Stopped-flow spectroscopy; Enzyme Mechanisms; Spectroscopy; Stopped-flow spectroscopy; Enzyme Catalysis; Nitrile hydratase; Iron; Kinetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.398909

Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe3+←S ligand-to-metal charge transfer band from 700 to 650 nm. The observed UV-visible absorption bands for the Fe3+-nitrile intermediate species are similar to low spin Fe3+-enzyme and model complexes bound by NO or N3−. These data provide spectroscopic evidence for the direct coordination of the nitrile substrate to the nitrile hydratase active site low spin Fe3+ center.
Background: No direct evidence exists for the direct coordination of nitrile to the Fe3+ active site in nitrile hydratases.
Results: The first Fe3+-nitrile intermediate species is reported using stopped-flow spectroscopy.
Conclusion: These data establish that the direct ligation of the nitrile substrate occurs during catalytic turnover.
Significance: Understanding the catalytic mechanism of nitrile hydratases is critical to harness their bioremediation and industrial potential.