Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism
Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect (kH/kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV–visible spectroscopy revealed a lactate-induced perturbation of the cofactor spectrum, notably increasing the absorbance at 340 nm, and demonstrated an interaction of the cofactor with the inhibitor sulfite. Here, a crystal structure of LarA provided greater resolution (2.4 Å) than previously reported and revealed sulfite binding to the pyridinium C4 atom of the reduced pincer cofactor, mimicking hydride reduction during a PCHT catalytic cycle. Finally, computational modeling supports hydride transfer to the cofactor at the C4 position or to the nickel atom, but with formation of a nickel-hydride species requiring dissociation of the His200 metal ligand. In aggregate, these studies provide compelling evidence that the nickel-pincer cofactor acts by a PCHT mechanism.
- Research Organization:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Organization:
- National Science Foundation (NSF)
- Grant/Contract Number:
- CHE-1516126; CHE-1654547
- OSTI ID:
- 1502215
- Journal Information:
- Biochemistry, Vol. 57, Issue 23; ISSN 0006-2960
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- ENGLISH
Web of Science
Functional Models of the Nickel Pincer Nucleotide Cofactor of Lactate Racemase
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journal | November 2019 |
Computational Insights into the Reaction Mechanisms of Nickel-Catalyzed Hydrofunctionalizations and Nickel-Dependent Enzymes
|
journal | March 2018 |
Functional Models of the Nickel Pincer Nucleotide Cofactor of Lactate Racemase
|
journal | November 2019 |
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