Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine.

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-l-tyrosine (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both l-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.

Cofactor Biogenesis in Cysteamine Dioxygenase: C-F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine.

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F2 -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and 19 F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink.

High-Frequency/High-Field Electron Paramagnetic Resonance and Theoretical Studies of Tryptophan-Based Radicals.

Tryptophan-based free radicals have been implicated in a myriad of catalytic and electron transfer reactions in biology. However, very few of them have been trapped so that biophysical characterizations can be performed in a high-precision context. In this work, tryptophan derivative-based radicals were studied by high-frequency/high-field electron paramagnetic resonance (HFEPR) and quantum chemical calculations. Radicals were generated at liquid nitrogen temperature with a photocatalyst, sacrificial oxidant, and violet laser. The precise g-anisotropies of l- and d-tryptophan, 5-hydroxytryptophan, 5-methoxytryptophan, 5-fluorotryptophan, and 7-hydroxytryptophan were measured directly by HFEPR. Quantum chemical calculations were conducted to predict both neutral and cationic radical spectra for comparison with the experimental data. The results indicate that under the experimental conditions, all radicals formed were cationic. Spin densities of the radicals were also calculated. The various line patterns and g-anisotropies observed by HFEPR can be understood in terms of spin-density populations and the positioning of oxygen atom substitution on the tryptophan ring. The results are considered in the light of the tryptophan and 7-hydroxytryptophan diradical found in the biosynthesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase.

Radical Trapping Study of the Relaxation of bis-Fe(IV) MauG.

The di-heme enzyme, MauG, utilizes a high-valent, charge-resonance stabilized bis-Fe(IV) state to perform protein radical-based catalytic chemistry. Though the bis-Fe(IV) species is able to oxidize remote tryptophan residues on its substrate protein, it does not rapidly oxidize its own residues in the absence of substrate. The slow return of bis-Fe(IV) MauG to its resting di-ferric state occurs via up to two intermediates, one of which has been previously proposed by Ma et al. (Biochem J 2016; 473:1769) to be a methionine-based radical in a recent study. In this work, we pursue intermediates involved in the return of high-valent MauG to its resting state in the absence of the substrate by EPR spectroscopy and radical trapping. The bis-Fe(IV) MauG is shown by EPR, HPLC, UV-Vis, and high-resolution mass spectrometry to oxidize the trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to a radical species directly. Nitrosobenzene was also employed as a trapping agent and was shown to form an adduct with high-valent MauG species. The effects of DMPO and nitrosobenzene on the kinetics of the return to di-ferric MauG were both investigated. This work eliminates the possibility that a MauG-based methionine radical species accumulates during the self-reduction of bis-Fe(IV) MauG.

Random-orientation high-spin electron spin resonance spectroscopy and comprehensive spectral analyses of the quintet dicarbene and dinitrene with meta-topological linkers: origins of peculiar line-broadening in fine-structure ESR spectra in organic rigid glasses.

In high-spin chemistry, random-orientation fine-structure (FS) electron spin resonance (ESR) spectroscopy entertains advantages as the most facile and convenient method to identify high-spin systems, as frequently reported in the literature. Random-orientation ESR spectroscopy applicable to organic high-spin entities can date back to the Wasserman and co-workers' attempt on the first spin-quintet dicarbene, m-phenylenebis(phenylmethlene) (m-PBPM), in the 2-MTHF glass in 1963 and 1967, following their successful work on randomly oriented triplet-state ESR spectroscopy. The FS ESR spectrum of m-PBPM in the 2-MTHF glass, however, has never fully been analyzed due to a peculiar line-broadening appearing at many canonical peaks. Organic high-spin spectra from most quintet dinitrenes also suffer from similar phenomena. Seemingly intrinsic line-diffusing or -broadening phenomena adversely affect the reliable determination of FS parameters for organic high-spin entities in rigid glasses. In high-spin chemistry, the line-broadening has been an obstacle that masks key FS transitions in many cases. Thus, both the origin of the broadening and the comprehensive spectral analysis have been a long-standing issue. In this report, we examine the origin of the line-broadening appearing in the FS ESR spectra, illustrated by a comprehensive spectral analysis for m-PBPM in the quintet ground state and the first-documented quintet-state dinitrene, m-phenylenebis(nitrene) (m-PBN) in the 2-MTHF glass. A complete analysis of the random-orientation FS spectra from m-PBPM diluted in the benzophenone crystal has shown that the g-anisotropy of m-PBPM is not prominent. Also the higher-order FS terms such as S(i)(2)S(j)(2) group-theoretically allowed for S = 2 are not necessary in spite of the argument for a hydrocarbon-based tetraradical (S = 2) in the ground state. Our new approach to the line-broadening analysis invokes both exact analytical solutions for the resonance fields of canonical peaks and the magnetic-parameters gradient method. The D- and E-values of m-PBPM acquired by the spectral simulation in this study give different molecular structures of the quintet dicarbene in the benzophenone crystal lattice (D = +0.0703(0) cm(-1), E = +0.0212(0) cm(-1)) and in the 2-MTHF glass (D = +0.0780(0) cm(-1), E = +0.0221(0) cm(-1)). Microscopic origins of the line-broadening observed for high-spin oligocarbenes or oligonitrenes generated by photolysis in organic glasses have been proposed.