Electron Transfer In Nitric Oxide Synthase Research Articles

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 functionalconformational 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 abi-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 ofrecombinant iNOS proteinalone. 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 statesof NOS proteins.

Oxygen activation in NO synthases: evidence for a direct role of the substrate.

Nitric oxide (NO) and the other reactive nitrogen species (RNOS) play crucial patho‐physiological roles at the interface of oxidative stress and signalling processes. In mammals, the NO synthases (NOSs) are the source of these reactive nitrogen species, and so to understand the precise biological role of RNOS and NO requires elucidation of the molecular functioning of NOS. Oxygen activation, which is at the core of NOS catalysis, involves a sophisticated sequence of electron and proton transfers. While electron transfer in NOS has received much attention, the proton transfer processes has been scarcely investigated. Here, we report an original approach that combines fast‐kinetic techniques coupled to resonance Raman spectroscopy with the use of synthetic analogues of NOS substrate. We characterise FeII‐O2 reaction intermediates in the presence of L‐arginine (Arg), alkyl‐ and aryl‐guanidines. The presence of new reaction intermediates, such as ferric haem‐peroxide, that was formerly postulated, was tracked by analysing the oxygen activation reaction at different times and with different excitation wavelengths. Our results suggest that Arg is not a proton donor, but indirectly intervenes in oxygen activation mechanism by modulating the distal H‐bond network and, in particular, by tuning the position and the role of the distal water molecule. This report supports a catalytic model with two proton transfers in step 1 (Arg hydroxylation) but only one proton transfer in step 2 (Nω‐hydroxy‐L‐arginine oxidation).

Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes

Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) ( Biochemistry 35, 8742–8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast k on constants at 4.5 °C: 6.6 × 10 8 M − 1 s − 1 for nNOS 726–749, 2.9 × 10 8 M − 1 s − 1 for eNOS 492–511 and 6.1 × 10 8 M − 1 s − 1 for iNOS 507–531, 3–4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca 2+ chelation with ETDA: 3.7 s − 1 for nNOS 726–749, 4.5 s − 1 for eNOS 492–511, and 0.063 s − 1 for iNOS 507–531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS 507–531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O(2) activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys(423), Lys(620), and Lys(660) were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys(423), Lys(620), and Lys(660), which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.

A Proline Residue in eNOS Hinge Region Restricts Inter‐Subunit Electron Transfer

The catalysis of nitric oxide by Nitric Oxide Synthase (NOS) involves electron transfer from the reductase (NOSr) to the heme in a NOS dimer. Among the NOS isoforms, eNOS shows the poorest activity. Why eNOS shows poor activity and heme reduction rate despite having a high flavin reduction rate remains an open question. The control element(s) responsible are also not completely identified. Our nNOSr crystal structure identified a 25-residue hinge that links the FMN module to the rest of the NOSr. We believe this hinge helps movement of the NOSr to mediate FMN module interaction with the heme for electron transfer. Sequence analysis revealed a proline in position 728 in eNOS hinge region corresponding to an isoleucine in the same position in nNOS. To check whether this proline residue compromised hinge flexibility to slow down interdomain electron transfer in eNOS compared to nNOS, we switched the proline with the isoleucine residue between eNOS (P728IeNOS) and nNOS (I958PnNOS). Compared to the wild-type, we observed a 33% increase in activity for P728IeNOS against a 48% drop in activity in I958PnNOS. Corresponding changes in heme reduction (38% increase for P728IeNOS and 27% decrease for I958PnNOS) as well as heme-NO build-up (42% increase for P728IeNOS and 53% decrease for I958PnNOS) rates for the two mutants also concurred with the activity changes although their flavin reduction rates did not change. Our study identifies the regulatory role of the hinge region in the intersubunit electron transfer in NOS for the first time opening avenues for further studies on the regulations differentiating the three NOS isoforms with respect to their catalytic behavior. This work was supported by NIH Grants GM51491 and CA53914 to D.J.S.

Structural Basis for Isozyme-specific Regulation of Electron Transfer in Nitric-oxide Synthase

Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme.

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

The idea that the gaseous freeradical molecule nitric oxide (NO) can regulate physiological processes has recently begun to achieve wide acceptance. However, many details of the mechanisms by which NO is synthesized, transported, and utilized as a messenger in neuronal and blood vessel function are not yet understood. This paper describes the cloning, characterization and functional expression of cDNAs encoding rat brain nitric oxide synthase (NOS), which is responsible for intracellular synthesis of NO from arginine. The NOS sequence provides a remarkable amount of information for predictions about the protein's function. The primary structure of the NOS polypeptide shows it to he a member of a class of electron transferases that have binding sites for the electron carriers NADPH, FAD and FMN. This strongly suggests that the mechanisms of electron transfer in NOS will be similar to those of the other members of this family. NOS is known to be regulated by a variety of factors, and the sequence reveals putative sites for interaction with calmodulin and for phosphorylation by cyclic AMP-dependent protein kinase. The authors also showed localization of NOS mRNA to NADPH diaphorase-enriched neurons, and propose that NOS activity may contribute to the relative resistance of these neurons to ischaemic and neurotoxic insults.