Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is an essential step in nitric oxide (NO) synthesis by NO synthase (NOS). The IET kinetics in neuronal and inducible NOS (nNOS and iNOS) holoenzymes have been previously determined in our laboratories by laser flash photolysis [reviewed in: C.J. Feng, G. Tollin, Dalton Trans., (2009) 6692-6700]. Here we report the kinetics of the IET in a bovine endothelial NOS (eNOS) holoenzyme in the presence and absence of added calmodulin (CaM). The IET rate constant in the presence of CaM is estimated to be ~ 4.3 s-1. No IET was observed in the absence of CaM, indicating that CaM is the primary factor in controlling the FMN–heme IET in the eNOS enzyme. The IET rate constant value for the eNOS holoenzyme is approximately 10 times smaller than those obtained for the iNOS and CaM-bound nNOS holoenzymes. Possible mechanisms underlying the difference in IET kinetics among the NOS isoforms are discussed. Because the rate-limiting step in the IET process in these enzymes is the conformational change from input state to output state, a slower conformational change (than in the other isoforms) is most likely to cause the slower IET in eNOS.

The rates of the bimolecular CO rebinding to the oxygenase domains of inducible and neuronal NOS proteins (iNOSoxy and nNOSoxy, respectively) after photolytic dissociation have been determined by laser flash photolysis. The following mutants at the isoform-specific sites (murine iNOSoxy N115L and rat nNOSoxy L337N, L337F) have been constructed to investigate role of the residues in the CO ligand accessibilities of the NOS isoforms. These residues are in the NOS distal substrate access channel. The effect of the (6R)-5,6,7,8-tetrahydrobiopterin (H4B) cofactor and L-arginine (Arg) substrate on the rates of CO rebinding have also been assessed. Addition of L-Arg to the iNOSoxy N115L mutant results in much faster CO rebinding rates, compared to the wild type. The results indicate that modifications to the iNOS channel in which the hydrophilic residue N115 is replaced by leucine (to resemble its nNOS cognate) open the channel somewhat, thereby improving access to the axial heme ligand binding position. On the other hand, introduction of a hydrophilic residue (L337N) or a bulky rigid aromatic residue (L337F) in the nNOS isoform does not significantly affect the kinetics profile, suggesting that the geometry of the substrate access pocket is not greatly altered. The bimolecular CO rebinding rate data indicate that the opening of the substrate access channel in the iNOS N115L mutant may be due to more widespread structural alterations induced by the mutation.

The FMN–heme intraprotein electron transfer (IET) kinetics in a human iNOS oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a calmodulin (CaM) construct that includes only its N-terminal globular domain, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly 4-fold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca2+ depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca2+-dependent reorganization of the NCaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of IET.

Sulfite oxidizing enzymes (SOEs) are molybdenum cofactor dependent enzymes that are found in plants, animals and bacteria. Sulfite oxidase (SO) is found in animals and plants, while sulfite dehydrogenase (SDH) is found in bacteria. In animals, SO catalyzes the oxidation of toxic sulfite to sulfate as the final step in the catabolism of the sulfur-containing amino acids, methionine and cysteine. In humans, sulfite oxidase deficiency is an inherited recessive disorder that produces severe neonatal neurological problems that lead to early death. Plant SO (PSO) also plays an important role in sulfite detoxification, but in addition serves as an intermediate enzyme in the assimilatory reduction of sulfate. In vertebrates the proposed catalytic mechanism of SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme. A similar mechanism is proposed for SDH, involving its molybdenum cofactor and c-type heme. However, PSO, which lacks an integral heme cofactor, uses molecular oxygen as its electron acceptor. Here we review recent results for SOEs from kinetic measurements, computational studies, electron paramagnetic resonance (EPR) spectroscopy, electrochemical measurements, and site-directed mutagenesis on active site residues of SOEs and of the flexible polypepetide tether that connects the heme and molybdenum domains of human SO. Rapid-kinetic studies of PSO are also discussed.

Sulfite oxidase (SO) is a vitally important molybdenum enzyme that catalyzes the oxidation of toxic sulfite to sulfate. The proposed catalytic mechanism of vertebrate SO involves two intramolecular one-electron transfer (IET) steps from the molybdenum cofactor to the iron of the integral b-type heme and two intermolecular one-electron steps to exogenous cytochrome c. In the crystal structure of chicken SO (Kisker et al., Cell, 1997, 91, 973–983), which is highly homologous to human SO (HSO), the heme iron and molybdenum centers are separated by 32 Å, and the domains containing these centers are linked by a flexible polypeptide tether. Conformational changes that bring these two centers into closer proximity have been proposed (Feng et al., Biochemistry, 2003, 41, 5816–21) to explain the relatively rapid IET kinetics, which are much faster than theoretically predicted from the crystal structure. In order to explore the proposed role(s) of the tether in facilitating this conformational change, both its length and flexibility were altered in HSO by site-specific mutagenesis and the reactivities of the resulting variants have been studied using laser flash photolysis and steady-state kinetics assays. Increasing the flexibility of the tether by mutating several conserved proline residues to alanines did not produce a discernable systematic trend in the kinetic parameters, although mutation of one residue (P105) to alanine produced a three-fold decrease in the IET rate constant. Deletions of non-conserved amino acids in the 14-residue tether, thereby shortening its length, resulted in more drastically reduced IET rate constants. Thus, the deletion of five amino acid residues decreased IET by 70-fold, so that it was rate-limiting in the overall reaction. The steady-state kinetic parameters were also significantly affected by these mutations, with the P111A mutation causing a five-fold increase in the sulfite Km value, perhaps reflecting a decrease in the ability to bind sulfite. The electron paramagnetic resonance spectra of these Proline to Alanine and deletion mutants are identical to those of wild type HSO, indicating no significant change in the Mo active site geometry.

There is still much that is unknown about how nitric oxide (NO) biosynthesis by NO synthase (NOS) isoform is tightly regulated at the molecular level. This is remarkable because impaired NO production in vivo has been implicated in an increasing number of diseases that currently lack effective treatments, including stroke and cancer. Given the significant public health burden of these diseases, the NOS enzyme family is a key target for development of new pharmaceuticals. Three NOS isoforms, inducible, endothelial and neuronal NOS (iNOS, eNOS and nNOS, respectively), achieve their key biological functions via intriguing regulations of interdomain electron transfer (IET) processes. Unlike iNOS, eNOS and nNOS isoforms are controlled by calmodulin (CaM) through facilitating catalytically significant IET processes. It is proposed that CaM activates NO synthesis in eNOS and nNOS through a conformational change of the flavin mononucleotide (FMN) domain from its shielded electron-accepting (input) state to a new electron-donating (output) state. The FMN–heme IET within the NOS output state is essential for NO synthesis at the catalytic heme. Due to lack of reliable techniques for specifically determining the inter-domain FMN–heme interactions and their direct effects on the catalytic heme center, the molecular mechanism that underlies the output state formation remains elusive. The recent developments in our understanding of mechanisms of the NOS output state formation that are driven by a combination of molecular biology, laser flash photolysis, and spectroscopic techniques are the subject of this perspective.

A procedure has been developed for directly depositing membrane fragments derived from bacterial (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (μ-opioid receptor- and MC4 receptor-transfected HEK cells, and rat trigeminal ganglion cells) onto the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c2 for the chromatophores, the peptide agonists DAMGO and Melanotan-II that are specific for the μ-opioid and MC4 receptors, and two non-peptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The KD values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.

All reported sulfite oxidizing enzymes have a conserved arginine in their active site which hydrogen bonds to the equatorial oxygen ligand on the Mo atom. Previous studies on the pathogenic R160Q mutant of human sulfite oxidase (HSO) have shown that Mo-heme intramolecular electron transfer (IET) is dramatically slowed when positive charge is lost at this position. In order to better understand the function that this conserved positively charged residue plays in IET, we have studied the equivalent uncharged substitutions, R55Q and R55M, as well as the positively charged substitution, R55K, in bacterial sulfite dehydrogenase (SDH). The heme and molybdenum cofactor (Moco) subunits are tightly associated in SDH, which makes it an ideal system for increasing the understanding of residue function in IET without the added complexity of the inter-domain movement that occurs in HSO. Unexpectedly, the uncharged SDH variants (R55Q and R55M) showed increased IET rate constants relative to the wildtype (3–4 fold) when studied by laser flash photolysis. The gain in function observed in SDHR55Q and SDHR55M suggests that the reduction of IET seen in HSOR160Q is not due to a required role of this residue in the IET pathway itself, but to the fact that it plays an important role in heme orientation during the inter-domain movement necessary for IET in HSO (as seen in viscosity experiments). The pH profiles of SDHwt, SDHR55M, and SDHR55Q show that the arginine substitution also alters the behavior of the Mo-heme IET equilibrium (Keq) and rate constants (ket) of both variants with respect to SDHWT enzyme. SDHWT has a ket that is independent of pH and a Keq that increases as pH decreases, whereas both SDHR55M and SDHR55Q have a ket that increases as pH decreases, and SDHR55M has a Keq that is pH independent. IET in the SDHR55Q variant is inhibited by sulfate in laser flash photolysis experiments, a behavior that differs from SDHWT, but which also occurs in HSO. IET in SDHR55K is slower than for SDHWT. A new analysis of the possible mechanistic pathways for sulfite oxidizing enzymes is presented and related to available kinetic and EPR results for these enzymes.

Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of ~150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 × 107 s−1. These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.

Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD = 0.2 ± 0.03 nM and 2 ± 0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB1 receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940- and WIN 55,212 - bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD = 27 ± 8 nM and KD = 10.7 ± 4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is essential in nitric oxide (NO) synthesis by NO synthase (NOS). Our previous laser flash photolysis studies provided a direct determination of the kinetics of the FMN–heme IET in a truncated oxyFMN construct of murine inducible NOS (iNOS), in which only the oxygenase and FMN domains along with the calmodulin (CaM) binding site are present [Feng et al. (2006) J. Am. Chem. Soc. 128, 3808-3811]. Here we report the kinetics of the IET in a human iNOS oxyFMN construct, a human iNOS holoenzyme and a murine iNOS holoenzyme, using CO photolysis in comparative studies on partially reduced NOS and a NOS oxygenase construct that lacks the FMN domain. The IET rate constants for the human and murine iNOS holoenzymes are 34 ± 5 s-1 and 35 ± 3 s-1, respectively, thereby providing a direct measurement of this IET between the catalytically significant redox couples of FMN and heme in the iNOS holoenzyme. These values are approximately an order of magnitude smaller than that in the corresponding iNOS oxyFMN construct, suggesting that in the holoenzyme the rate-limiting step in the IET is the conversion of the shielded electron-accepting (input) state to a new electron-donating (output) state. The fact that there is no rapid IET component in the kinetic traces obtained with the iNOS holoenzyme implies that the enzyme remains mainly in the input state. The IET rate constant value for the iNOS holoenzyme is similar to that obtained for a CaM-bound neuronal NOS (nNOS) holoenzyme, suggesting that CaM activation effectively removes the inhibitory effect of the unique autoregulatory insert in nNOS.

Comparative CO photolysis kinetics studies on wild type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN–heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that has been developed in our laboratories and applied to the study of membrane-associated proteins, especially GPCRs. It has high sensitivity and requires no labeling of materials, and can monitor changes in proteolipid mass density and conformation in real time using plasmon excitation by light polarized both perpendicular and parallel to the resonator surface. Direct measurements will be described of the association of ligands and G-proteins to GPCRs incorporated into a self-assembled lipid bilayer deposited on the silica surface of a PWR resonator. These studies have provided new insights into the functioning of this important class of signaling proteins.

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand–receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (Kd = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.

Sulfite oxidizing enzymes are essential mononuclear molybdenum (Mo) proteins involved in sulfur metabolism of animals, plants and bacteria. There are three such enzymes presently known: (1) sulfite oxidase (SO) in animals, (2) SO in plants, and (3) sulfite dehydrogenase (SDH) in bacteria. X-ray crystal structures of enzymes from all three sources (chicken SO, Arabidopsis thaliana SO, and Starkeya novella SDH) show nearly identical square pyramidal coordination around the Mo atom, even though the overall structures of the proteins and the presence of additional cofactors vary. This structural information provides a molecular basis for studying the role of specific amino acids in catalysis. Animal SO catalyzes the final step in the degradation of sulfur-containing amino acids and is critical in detoxifying excess sulfite. Human SO deficiency is a fatal genetic disorder that leads to early death, and impaired SO activity is implicated in sulfite neurotoxicity. Animal SO and bacterial SDH contain both Mo and heme domains, whereas plant SO only has the Mo domain. Intraprotein electron transfer (IET) between the Mo and Fe centers in animal SO and bacterial SDH is a key step in the catalysis, which can be studied by laser flash photolysis in the presence of deazariboflavin. IET studies on animal SO and bacterial SDH clearly demonstrate the similarities and differences between these two types of sulfite oxidizing enzymes. Conformational change is involved in the IET of animal SO, in which electrostatic interactions may play a major role in guiding the docking of the heme domain to the Mo domain prior to electron transfer. In contrast, IET measurements for SDH demonstrate that IET occurs directly through the protein medium, which is distinctly different from that in animal SO. Point mutations in human SO can result in significantly impaired IET or no IET, thus rationalizing their fatal effects. The recent developments in our understanding of sulfite oxidizing enzyme mechanisms that are driven by a combination of molecular biology, rapid kinetics, pulsed electron paramagnetic resonance (EPR), and computational techniques are the subject of this review.

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.

Increasing evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions having resistance to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In the present studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR; a G-protein coupled receptor, GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor incorporated preferentially into the POPC-rich domain; when an agonist or an antagonist was bound, the receptor incorporated preferentially into the SM-rich component, although with a two-fold greater propensity for this microdomain in the case of the agonist. G-protein binding to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than to the receptor in the PC-rich domain. The binding of agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich into the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor elongates upon agonist-activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component.