Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants.

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSµ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSµ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.

Probing Protein Dynamics in Neuronal Nitric Oxide Synthase by Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) is responsible for the biosynthesis of nitric oxide (NO), an important signaling molecule controlling diverse physiological processes such as neurotransmission and vasodilation. Neuronal NOS (nNOS) is a calmodulin (CaM)-controlled enzyme. In the absence of CaM, several intrinsic control elements, along with NADP+ binding, suppress electron transfer across the NOS domains. CaM binding relieves the inhibitory factors to promote the electron transport required for NO production. The regulatory dynamics of nNOS control elements are critical to governing NO signaling, yet mechanistic questions remain, because the intrinsic dynamics of NOS thwart traditional structural biology approaches. Here, we have employed cross-linking mass spectrometry (XL MS) to probe regulatory dynamics in nNOS, focusing on the CaM-responsive control elements. Quantitative XL MS revealed conformational changes differentiating the nNOS reductase (nNOSred) alone, nNOSred with NADP+, nNOS-CaM, and nNOS-CaM with NADP+. We observed distinct effects of CaM vs NADP+ on cross-linking patterns in nNOSred. CaM induces striking global changes, while the impact of NADP+ is primarily localized to the NADPH-binding subdomain. Moreover, CaM increases the abundance of intra-nNOS cross-links that are related to the formation of the inter-CaM-nNOS cross-links. Taken together, these XL MS results demonstrate that CaM and NADP+ site-specifically alter the nNOS conformational landscape.

Interdomain Interactions Modulate the Active Site Dynamics of Human Inducible Nitric Oxide Synthase.

Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.

A dye-andrographolide assembly as a turn-on sensor for detection of phthalate in both cells and fish.

Phthalates can penetrate the environment and enrich various aquatic organisms through the food chain, which is involved in promoting the growth of breast cancer. It is of current interest to develop new sensors for phthalates. We herein reported a hydrogen-bond competing fluorescent sensor, BANP, for the detection of dibutyl phthalate (DBP). The BANP compound was synthesized by assembling andrographolide (Andro), nitro- and cyano-substituted BODIPY dye (BCN), and polyethylene glycol derivatives (DSPE-mPEG5000). BANP was found to be a turn-on fluorescent probe for DBP in water with a detection limit of 0.13 µg/g; the DBP-water system acts as a hydrogen bond switch to turn on the fluorescence. And BANP fluorescently detected DBP in contaminated fish meat. Moreover, BANP sensed the DBP-induced growth of human breast cancer MCF-7 cells, and the release of Andro in the DBP-cultivated cancer cells inhibited the proliferation of the MCF-7 cells. Taken together, BANP is a DBP-responsive probe for sensitive DBP detection in water, cells, and fish meats. The BANP sensor may be used in both in vitro fluorescence and cellular imaging analyses. Our results show that guest-induced reassembly brings forth significant fluorescence change, which is a promising way of designing new fluorescent probes for the analysis of phthalates in the environment and food.

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases.

Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90ß contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSµ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSµ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSµ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSµ and nNOSα proteins.

An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase.

Intraprotein interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain-domain alignment (J Inorg Biochem 153:186-196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flash photolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitory effect was abolished by a single L406 → E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.

Heat shock protein 90 enhances the electron transfer between the FMN and heme cofactors in neuronal nitric oxide synthase.

Heat shock protein 90 (Hsp90) is a key regulator of nitric oxide synthase (NOS) in vivo. Despite its functional importance, little is known about the underlying molecular mechanism. Here, purified dimeric human Hsp90α was used to investigate whether (and if so, how) Hsp90 affects the FMN-heme interdomain electron transfer (IET) step in NOS. Hsp90α increases the IET rate for rat neuronal NOS (nNOS) in a dose-saturable manner, and a single charge-neutralization mutation at conserved Hsp90 K585 abolishes the effect. The kinetic results with added Ficoll 70, a crowder, further indicate that Hsp90 enhances the FMN-heme IET through specific association with nNOS. The Hsp90-nNOS docking models provide hints on the putative role of Hsp90 in constraining the available conformational space for the FMN domain motions.

Mitochondrial targeting nano-curcumin for attenuation on PKM2 and FASN.

Tumor cells are sensitive to the disturbance of mitochondrial functions. Attenuation of dysfunctional mitochondria by natural compounds is an emerging strategy for the recovery of abnormal energy metabolism of cancer. To develop a nano-sized curcumin (CUR) in attenuating the energy metabolism of cancer cells, herein, a coral-shaped nano-transporter DNA-FeS2-DA nanoparticle was synthesized using double-stranded DNA rich in 'GAG' and 'GC' series as a template and poly-dopamine as an adhesive. CUR was successfully loaded to DNA-FeS2-DA with a molar ratio of ssDNA: CUR of 1:16, forming CUR@DNA-FeS2-DA. This nano-curcumin can readily enter mitochondrion in MCF-7 cancer cells. The CUR@DNA-FeS2-DA nanocomposite displays desirable photothermal effect and stability, while its CUR can be released gradually in the weak acid environment. The expression of both pyruvate kinase M2 and fatty acid synthase in the MCF-7 cancer cells were noticeably inhibited by CUR@DNA-FeS2-DA. Given the controlled release and mitochondria-targeting properties, this CUR@DNA-FeS2-DA nanocomposite is a promising new drug entity for intervening the energy metabolism of cancer cells.

Positional Distributions of the Tethered Modules in Nitric Oxide Synthase: Monte Carlo Calculations and Pulsed EPR Measurements.

The nitric oxide synthase (NOS) enzyme consists of multiple domains connected by flexible random coil tethers. In a catalytic cycle, the NOS domains move within the limits determined by the length and flexibility of the interdomain tethers and form docking complexes with each other. This process represents a key component of the electron transport from the flavin adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate binding domain to the catalytic heme centers located in the oxygenase domain. Studying the conformational behavior of NOS is therefore imperative for a full understanding of the overall catalytic mechanism. In this work, we have investigated the equilibrium positional distributions of the NOS domains and the bound calmodulin (CaM) by using Monte Carlo calculations of the NOS conformations. As a main experimental reference, we have used the magnetic dipole interaction between a bifunctional spin label attached to T34C/S38C mutant CaM and the NOS heme centers, which was measured by pulsed electron paramagnetic resonance. In general, the calculations of the conformational distributions allow one to determine the range and statistics of positions occupied by the tethered protein domains, assess the crowding effect of the multiple domains on each other, evaluate the accessibility of various potential domain docking sites, and estimate the interaction energies required to achieve target populations of the docked states. In the particular application described here, we have established the specific mechanisms by which the bound CaM facilitates the flavin mononucleotide (FMN)/heme interdomain docking in NOS. We have also shown that the intersubunit FMN/heme domain docking and electron transfer in the homodimeric NOS protein are dictated by the existing structural makeup of the protein. Finally, from comparison of the calculated and experimental docking probabilities, the characteristic stabilization energies for the CaM/heme domain and the FMN domain/heme domain docking complexes have been estimated as -4.5kT and -10.5kT, respectively.

Effect of Macromolecular Crowding on the FMN-Heme Intraprotein Electron Transfer in Inducible NO Synthase.

Previous biochemical studies of nitric oxide synthase enzymes (NOSs) were conducted in diluted solutions. However, the intracellular milieu where the proteins perform their biological functions is crowded with macromolecules. The effect of crowding on the electron transfer kinetics of multidomain proteins is much less understood. Herein, we investigated the effect of macromolecular crowding on the FMN-heme intraprotein interdomain electron transfer (IET), an obligatory step in NOS catalysis. A noticeable increase in the IET rate in the bidomain oxygenase/FMN (oxyFMN) and the holoprotein of human inducible NOS (iNOS) was observed upon addition of Ficoll 70 in a nonsaturable manner. Additionally, the magnitude of IET enhancement for the holoenzyme is much higher than that that of the oxyFMN construct. The crowding effect is also evident at different ionic strengths. Importantly, the enhancing extent is similar for the iNOS oxyFMN protein with added Ficoll 70 and Dextran 70 that give the same solution viscosity, showing that specific interactions do not exist between the NOS protein and the crowder. Moreover, the population of the docked FMN-heme state is significantly increased upon addition of Ficoll 70 and the fluorescence lifetime values do not correspond to those in the absence of Ficoll 70. The steady-state cytochrome c reduction by the holoenzyme is noticeably enhanced by the crowder, while the ferricyanide reduction is unchanged. The NO production activity of the iNOS holoenzyme is stimulated by Ficoll 70. The effect of macromolecular crowding on the kinetics can be rationalized on the basis of the excluded volume effect, with an entropic origin. The intraprotein electron transfer kinetics, fluorescence lifetime, and steady-state enzymatic activity results indicate that macromolecular crowding modulates the NOS electron transfer through multiple pathways. Such a mechanism should be applicable to electron transfer in other multidomain redox proteins.

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme.

Phosphorylation is an important pathway for the regulation of nitric oxide synthase (NOS) at the posttranslational level. However, the molecular underpinnings of NOS regulation by phosphorylations remain unclear to date, mainly because of the problems in making a good amount of active phospho-NOS proteins. Herein, we have established a system in which recombinant rat nNOS holoprotein can be produced with site-specific incorporation of phosphoserine (pSer) at residue 1412, using a specialized bacterial host strain for pSer incorporation. The pSer1412 nNOS protein demonstrates UV-Vis, far-UV CD and fluorescence spectral properties that are identical to those of nNOS overexpressed in other bacterial strains. The protein is also functional, possessing normal NO production and NADPH oxidation activities in the presence of abundant substrate L-Arg. Conversely, the rate of FMN-heme interdomain electron transfer (IET) in pSer1412 nNOS is considerably lower than that of wild-type (wt) nNOS, while the phosphomimetic S1142E mutant possesses similar electron transfer kinetics to that of wt. The successful incorporation and high yield of pSer1412 into rat nNOS and the significant change in the IET kinetics upon the phosphorylation demonstrate a highly useful method for incorporating native phosphorylation sites as a substantial improvement to commonly used phosphomimetics.

Ground State Nuclear Magnetic Resonance Chemical Shifts Predict Charge-Separated Excited State Lifetimes.

Dichalcogenolene platinum(II) diimine complexes, (LE,E')Pt(bpy), are characterized by charge-separated dichalcogenolene donor (LE,E') → diimine acceptor (bpy) ligand-to-ligand charge transfer (LL'CT) excited states that lead to their interesting photophysics and potential use in solar energy conversion applications. Despite the intense interest in these complexes, the chalcogen dependence on the lifetime of the triplet LL'CT excited state remains unexplained. Three new (LE,E')Pt(bpy) complexes with mixed chalcogen donors exhibit decay rates that are dominated by a spin-orbit mediated nonradiative pathway, the magnitude of which is proportional to the anisotropic covalency provided by the mixed-chalcogen donor ligand environment. This anisotropic covalency is dramatically revealed in the 13C NMR chemical shifts of the donor carbons that bear the chalcogens and is further probed by S K-edge XAS. Remarkably, the NMR chemical shift differences also correlate with the spin-orbit matrix element that connects the triplet excited state with the ground state. Consequently, triplet LL'CT excited state lifetimes are proportional to both functions, demonstrating that specific ground state NMR chemical shifts can be used to evaluate spin-orbit coupling contributions to excited state lifetimes.

Role of an isoform-specific residue at the calmodulin-heme (NO synthase) interface in the FMN - heme electron transfer.

The interface between calmodulin (CaM) and the NO synthase (NOS) heme domain is the least characterized interprotein interface that the NOS isoforms must traverse through during catalysis. Our previous molecular dynamics simulations predicted a salt bridge between K497 in human inducible NOS (iNOS) heme domain and D118(CaM). Herein, the FMN - heme interdomain electron transfer (IET) rate was found to be notably decreased by charge-reversal mutation, while the IET in the iNOS K497D mutant is significantly restored by the CaM D118K mutation. The results of wild-type protein with added synthetic peptides further demonstrate the critical nature of K497 relative to the rest of the peptide sequence in modulating the IET. These data provide definitive evidence supporting the regulatory role of the isoform-specific K497 residue.

A docked state conformational dynamics model to explain the ionic strength dependence of FMN - heme electron transfer in nitric oxide synthase.

The FMN-heme interdomain electron transfer (IET) in nitric oxide synthase (NOS) is a key stage of the electron transport chain, which supplies the catalytic heme site(s) with the NADPH-derived electrons. While there is a recognition that this IET depends on both the electron tunneling and the conformational dynamics, the detailed mechanism remains unclear. In this work, the IET kinetics were measured by laser flash photolysis for a bidomain oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS) over the ionic strength range from 0.1 to 0.5 M. The forward (heme → FMN, kETf) and backward (FMN → heme, kETb) intrinsic IET rate constants were determined from the analysis of the observed IET rates using the additional information regarding the conformational dynamics obtained from the FMN fluorescence lifetime measurements and theoretical estimates. Both kETf and kETb exhibit a bell-shaped dependence on the ionic strength, I, with the maximum rates corresponding to I ~ 0.2 M. This dependence was explained using a new model, which considers the effect of formation of pairs between the protein surface charged residues and solution ions on the docked state dynamics. The trial simulations of the intrinsic IET rate dependences using this model show that the data can be reproduced using reasonable energetic, structural, and chemical parameters. The suggested model can explain both the monophasic and biphasic ionic strength dependences and can be used to rationalize the interprotein/interdomain electron transfer rates for other types of protein systems where the docked state is sufficiently long-lived.

Deciphering mechanism of conformationally controlled electron transfer in nitric oxide synthases.

Electron transfer is a fundamental process in life that is very often coupled to catalysis within redox enzymes through a stringent control of protein conformational movements. Mammalian nitric oxide synthase (NOS) proteins are redox flavo-hemoproteins consisting of multiple modular domains. The NOS enzyme is exquisitely regulated in vivo by its partner, the Ca2+ sensing protein calmodulin (CaM), to control production of nitric oxide (NO). The importance of functional domain motion in NOS regulation has been increasingly recognized. The significant size and flexibility of NOS is a tremendous challenge to the mechanistic studies. Herein recent applications of modern biophysical techniques to NOS problems have been critically analyzed. It is important to note that any current biophysical technique alone can only probe partial aspects of the conformational dynamics due to limitations in the technique itself and/or the sample preparations. It is necessary to combine the latest methods to comprehensively quantitate the key conformational aspects (conformational states and distribution, conformational change rates, and domain interacting interfaces) governing the electron transfer. This is to answer long-standing central questions about the NOS isoforms by defining how specific CaM-NOS interactions and regulatory elements underpin the distinct conformational behavior of the NOS isoform, which in turn determine unique electron transfer and NO synthesis properties. This review is not intended as comprehensive, but as a discussion of prospects that promise impact on important questions in the NOS enzymology field.

Metastatic melanoma imaging using a novel Tc-99m-labeled lactam-cyclized alpha-MSH peptide.

The purpose of this study was to determine the metastatic melanoma imaging property of 99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSHhex {hydrazinonicotinamide-8-aminooctanoic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}. HYNIC-Aoc-Nle-CycMSHhex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The IC50 value of HYNIC-Aoc-Nle-CycMSHhex was 0.78 ±â€¯0.13 nM for B16/F10 melanoma cells. 99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSHhex displayed significantly higher uptake (14.26 ±â€¯2.74 and 10.45 ±â€¯2.31% ID/g) in B16/F10 metastatic melanoma-bearing lung than that in normal lung (0.90 ±â€¯0.15 and 0.53 ±â€¯0.14% ID/g) at 2 and 4 h post-injection, respectively. B16/F10 pulmonary metastatic melanoma lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-Aoc-Nle-CycMSHhex as an imaging probe at 2 h post-injection, underscoring its potential as an imaging probe for metastatic melanoma detection.

Evaluation of novel 111In-labeled gonadotropin-releasing hormone peptides for human prostate cancer imaging.

The purpose of this study was to evaluate the tumor targeting and imaging properties of novel 111In-labeled gonadotropin-releasing hormone (GnRH) peptides for human prostate cancer. Three new 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-linker-d-Phe-(d-Lys6-GnRH) peptides with different hydrocarbon linkers were designed to evaluate their effects on GnRH receptor binding affinities. The Aoc (aminooctanoic acid) linker was better than ßAla (3-aminopropanoic acid) and Aun (aminoundecanoic acid) linkers in retaining strong receptor binding affinity. DOTA-Aoc-d-Phe-(d-Lys6-GnRH) exhibited 6.6±0.1nM GnRH receptor binding affinity. 111In-DOTA-Aoc-d-Phe-(d-Lys6-GnRH) exhibited fast tumor uptake and urinary clearance in DU145 human prostate cancer-xenografted nude mice. The DU145 tumor lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using 111In-DOTA-Aoc-d-Phe-(d-Lys6-GnRH) as an imaging probe, providing an insight into the design of new GnRH peptides for prostate cancer in the future.

Exploring second coordination sphere effects in nitric oxide synthase.

Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) → Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.

Role of a Conserved Tyrosine Residue in the FMN-Heme Interdomain Electron Transfer in Inducible Nitric Oxide Synthase.

The interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains is essential in the biosynthesis of nitric oxide (NO) by the NO synthase (NOS) enzymes. A conserved tyrosine residue in the FMN domain (Y631 in human inducible NOS) was proposed to be a key part of the electron transfer pathway in the FMN/heme docked complex model. In the present study, the FMN-heme IET kinetics in the Y631F mutant and wild type of a bidomain oxygenase/FMN construct of human inducible NOS were determined by laser flash photolysis. The rate constant of the Y631F mutant is significantly decreased by ∼75% (compared to the wild type), showing that the tyrosine residue indeed facilitates the FMN-heme IET through the protein medium. The IET rate constant of the wild type protein decreases from 345 to 242 s-1 on going from H2O to 95% D2O, giving a solvent kinetic isotope effect of 1.4. In contrast, no deuterium isotope effect was observed for the Tyr-to-Phe mutant. Moreover, an appreciable change in the wild type iNOS IET rate constant value was observed upon changing pH. These results indicate that the FMN-heme IET is proton coupled, in which the conserved tyrosine residue may play an important role.