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Lactate Racemase Nickel-Pincer Cofactor Operates by a Proton-Coupled Hydride Transfer Mechanism

Journal Article · · Biochemistry
 [1];  [2];  [1];  [3];  [1];  [1];  [2];  [1]
  1. Michigan State Univ., East Lansing, MI (United States)
  2. Univ. of Nevada, Reno, NV (United States)
  3. Univ. catholique de Louvain, Louvain-La-Neuve (Belgium)
+ Show Author Affiliations
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
OSTI ID:
1502215
Journal Information:
Biochemistry, Journal Name: Biochemistry Journal Issue: 23 Vol. 57; ISSN 0006-2960
Publisher:
American Chemical Society (ACS)Copyright Statement
Country of Publication:
United States
Language:
ENGLISH

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Cited By (3)

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
Functional Models of the Nickel Pincer Nucleotide Cofactor of Lactate Racemase journal November 2019

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
anions
cations
hydride transfer
nickel
peptides and proteins