title: 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.
authors.affiliation: Institute of Materials Science, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan. tnoguchi@ims.tsukuba.ac.jp
authors.forename: Takumi
authors.initials: T
authors.lastname: Noguchi
authors.forename: Masaki
authors.initials: M
authors.lastname: Nojiri
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Takei
authors.forename: Masafumi
authors.initials: M
authors.lastname: Odaka
authors.forename: Nobuo
authors.initials: N
authors.lastname: Kamiya
mesh_terms: D003545:Cysteine / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D008027:Light; D011522:Protons; D012240:Rhodococcus / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013434:Sulfenic Acids / Q000737:chemistry*; D013441:Sulfinic Acids / Q000737:chemistry*; D013460:Sulfur Isotopes / Q000378:metabolism; D014732:Vibration
chemical_list: D011522:Protons; D013434:Sulfenic Acids; D013441:Sulfinic Acids; D013460:Sulfur Isotopes; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine