The NOS ouput state for NO production is a complex of the FMN-binding domain and heme domain, and thereby it faciltates the interdomain electron transfer from the FMN to the catalytic heme site. Emerging evidence suggests that interdomain FMN/heme interactions are important in formation of the output state by guiding the docking of the FMN domain to the heme domain. In this study, notable effects of mutations in the adjacent FMN domain on the heme structure in a human iNOS bi-domain oxygnease/FMN construct have been observed by using low-temperature MCD spectroscopy. The comparative MCD study of wild type and mutant proteins clearly indicate that a properly docked FMN domain contributes to the observed L-Arg-perturbation of heme MCD spectrum in the wild type protein, and that the conserved surface residues at the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.In mammals, nitric oxide (NO) is synthesized by nitric oxide synthase (NOS), a homodimeric flavo-hemoprotein that catalyzes the oxidation of L-arginine (Arg) to NO and L-citrulline with NADPH and O 2 as co-substrates. 1 Each NOS subunit contains a C-terminal electron-supplying reductase unit with binding sites for NADPH (electron source), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), and an N-terminal catalytic heme-containing oxygenase domain. The FMN domain and oxygenase domain are linked by a calmodulin (CaM) binding region. Three NOS isoforms, iNOS, eNOS and nNOS, achieve their key biological mkirk@unm.edu, cfeng@salud.unm.edu. Wild type (wt) human iNOS oxyFMN construct was expressed and purified as described elsewhere. 10 Located at the edge of the FMN domain of human iNOS, E546 and E603 are charged surface residues that are conserved in all NOS isoforms (Fig. S1, Supporting Information). In order to address the roles of these charged residues in interdomain FMN/heme interactions, E546N and E603N mutants were constructed, expressed, and purified (see Supporting Information). The MCD samples were prepared by using microvolumetric techniques, and temperature dependent MCD spectra, arising from the ferric heme site, were obtained at 5K, 10K and 20K in a 7T applied magnetic field (Fig. 2). 11 Importantly, the MCD spectrum of the wt protein is noticeably perturbed upon incubation with the substrate L-Arg (Fig. 2a), whereas the E546N and E603N mutants have similar MCD spectra in both the presence and absence of L-Arg (Fig. 2b, and Fig. S5 in Supporting Information, respectively). Previous room temperature MCD studies on wt nNOS holoenzyme also suggested a perturbation of the heme signal upon L-Arg binding, 12 consistent with our low-temperature observations for the wt iNOS oxyFMN construct, vide infra. This L-Arg perturbation in the nNOS holoenzyme has previously been attributed to the conversion of a mixed high-spin/lowspin state to an exclusively high-spin heme when L-Arg binds near the heme site. 12 We have probed the ferric heme spin states in the wt and mu...
A detailed EPR and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed which serves as a basis for developing a valence bond description of C-H activation and transition state stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde Inhibited XO have been analyzed in order to provide information regarding the relationship between the g-, 95,97Mo hyperfine (AMo), and the 13C hyperfine (AC) tensors. The analysis of the EPR spectra have allowed for greater insight into the electronic origin of key delocalizations within the Mo-Oeq-C fragment, and how these contribute to C-H bond activation/cleavage and transition state (TS) stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo=S π→C-H σ* (ΔE= 24.3 kcal/mol) and C-H σ → Mo=S π* (ΔE = 20.0 kcal/mol) back donation are important in activating the substrate for C-H bond for cleavage. Additional contributions to C-H activation derive from Oeq lp→C-H σ* (lp = lone pair; ΔE = 8.2 kcal/mol), and S lp→C-H σ* (ΔE = 13.2 kcal/mol) stabilizing interactions. The Oeq donor ligand that derives from water is part of the Mo-Oeq-C fragment probed in the EPR spectra of XO Inhibited, and the observation of Oeq lp→C-H σ* back donation indicates a key role for the Oeq in activating the substrate C-H bond for cleavage. We also show that the Oeq donor plays an even more important role in transition state (TS) stabilization. We find that Oeq→(Mo + C) charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal/mol) and the Mo-Oeq-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-Oeq-C delocalization at the TS allows for the TS to be described in valence bond terms as a resonance hybrid of the reactant (R) and product (P) valence bond wavefunctions.
Electronic paramagnetic resonance, electronic absorption, and magnetic circular dichroism spectroscopies have been performed on YedY, a SUOX fold protein with a Mo domain that is remarkably similar to that found in chicken sulfite oxidase, A. thaliana plant sulfite oxidase, and the bacterial sulfite dehydrogenase from S. novella. Low-energy dithiolene→Mo and cysteine thiolate→Mo charge transfer bands have been assigned for the first time in a Mo(V) form of a SUOX fold protein, and the spectroscopic data have been used to interpret the results of bonding calculations. The analysis shows that second coordination sphere effects modulate dithiolene and cysteine sulfur covalency contributions to the Mo bonding scheme. Namely, a more acute Ooxo-Mo-SCys-C dihedral angle results in increased cysteine thiolate S→Mo charge transfer and a high g1 in the EPR spectrum. The spectrosocopic results, coupled with the available structural data, indicate that these second coordination sphere effects may play key roles in modulating the active site redox potential, facilitating hole superexchange pathways for electron transfer regeneration, and affecting the type of reactions catalyzed by sulfite oxidase family enzymes.
We have obtained low temperature MCD spectra for ferric cyano complexes of the wild type and E546N mutant of a human iNOS oxygenase/FMN construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the L-arginine-bound ferric iNOS heme [Sempombe et al., J. Am. Chem. Soc. 2009, 131, 6940–6941]. Addition of L-arginine to the wild type protein causes notable changes in the CN−-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with L-arginine is absent in the CN− complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. EPR spectroscopy also reveals a substrate dependency of the spectra. These results indicate that the interdomain FMN–heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.
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