Protonation Structures of Cys-Sulfinic and Cys-Sulfenic Acids in the Photosensitive Nitrile Hydratase Revealed by Fourier Transform Infrared Spectroscopy

Abstract

Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.

Catalytic Mechanism of Nitrile Hydratase Proposed by Time-resolved X-ray Crystallography Using a Novel Substrate, tert-Butylisonitrile

“…All ligand residues are involved in a strictly conserved motif of the ␣ subunit, Cys 1 -Xaa-Leu-Cys 2 -Ser-Cys 3 , where two amide nitrogens of Ser and Cys 3 and three Cys sulfurs are coordinated to the metal (6). Cys 2 and Cys 3 are post-translationally modified to cysteine-sulfinic acid and cysteine-sulfenic acid, respectively (7), which probably take deprotonated forms at the metal site (11). The sixth ligand site is occupied by a solvent molecule (8) or by a NO molecule in nitrosylated iron-type NHase (7).…”

Regulation of Peroxiredoxins by Nitric Oxide in Immunostimulated Macrophages

“…Based on the notion than NO induces cysteine sulfinylation in vitro (27,28), we evaluated whether NO would also promote overoxidation of Prxs. Using an antibody that immunoreacts with the sulfinylated form of the four 2-Cys Prxs (I-IV), we thus monitored the level of overoxidized Prxs in macrophages that were stimulated to produce NO ( Fig.…”

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

“…3b However, aside from FT-IR, there are no direct spectroscopic probes of the CysS − ligand oxidation states in the active form of the enzyme. 10 Although active sites having transition-metal-bound oxidized thiolate residues are rare in nature, mutational studies on these thiolate residues have shown them to be catalytically relevant. 11,12 The catalytic mechanism of this site is not well understood, and there are several proposals regarding the role of the low-spin Fe III center in activating nitriles for hydrolysis.…”