Neurogranin modulates the rate of association between calmodulin and target peptides.

The best-known mode of action of calmodulin (CaM) is binding of Ca2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca2+, including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca2+-dependent CaM targets at equilibrium because they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca2+-dependent targets. This mode of regulation may be domain specific because PEP-19 binds to the C-domain of CaM, whereas Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca2+-CaM at low levels of free Ca2+ (0.25-1.5 µM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca2+. We also show that Ng greatly decreases the rate of association of Ca2+-CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca2+ oscillations.

Partial agonism in heteromeric GLUK2/GLUK5 kainate receptor.

Kainate receptors are a subtype of ionotropic glutamate receptors that form transmembrane channels upon binding glutamate. Here, we have investigated the mechanism of partial agonism in heteromeric GluK2/K5 receptors, where the GluK2 and GluK5 subunits have distinct agonist binding profiles. Using single-molecule Förster resonance energy transfer, we found that at the bi-lobed agonist-binding domain, the partial agonist AMPA-bound receptor occupied intermediate cleft closure conformational states at the GluK2 cleft, compared to the more open cleft conformations in apo form and more closed cleft conformations in the full agonist glutamate-bound form. In contrast, there is no significant difference in cleft closure states at the GluK5 agonist-binding domain between the partial agonist AMPA- and full agonist glutamate-bound states. Additionally, unlike the glutamate-bound state, the dimer interface at the agonist-binding domain is not decoupled in the AMPA-bound state. Our findings suggest that partial agonism observed with AMPA binding is mediated primarily due to differences in the GluK2 subunit, highlighting the distinct contributions of the subunits towards activation.

Conformational spread and dynamics in allostery of NMDA receptors.

Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.

Allosteric Changes in the NMDA Receptor Associated with Calcium-Dependent Inactivation.

N-methyl-D-aspartate (NMDA) receptors mediate synaptic excitatory signaling in the mammalian central nervous system by forming calcium-permeable transmembrane channels upon binding glutamate and coagonist glycine. Ca2+ influx through NMDA receptors leads to channel inactivation through a process mediated by resident calmodulin bound to the intracellular C-terminal segment of the GluN1 subunit of the receptor. Using single-molecule FRET investigations, we show that in the presence of calcium-calmodulin, the distance across the two GluN1 subunits at the entrance of the first transmembrane segment is shorter and the bilobed cleft of the glycine-binding domain in GluN1 is more closed when bound to glycine and glutamate relative to what is observed in the presence of barium-calmodulin. Consistent with these observations, the glycine deactivation rate is slower in the presence of calcium-calmodulin. Taken together, these results show that the binding of calcium-calmodulin to the C-terminus has long-range allosteric effects on the extracellular segments of the receptor that may contribute to the calcium-dependent inactivation.

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Six-Transmembrane Epithelial Antigen of Prostate 1 (STEAP1) Has a Single b Heme and Is Capable of Reducing Metal Ion Complexes and Oxygen.

STEAP1, six-transmembrane epithelial antigen of prostate member 1, is strongly expressed in several types of cancer cells, particularly in prostate cancer, and inhibition of its expression reduces the rate of tumor cell proliferation. However, the physiological function of STEAP1 remains unknown. Here for the first time, we purified a mammalian (rabbit) STEAP1 at a milligram level, permitting its high-quality biochemical and biophysical characterizations. We found that STEAP1 likely assembles as a homotrimer and forms a heterotrimer when co-expressed with STEAP2. Each STEAP1 protomer binds one heme prosthetic group that is mainly low-spin with a pair of histidine axial ligands, with small portions of high-spin and P450-type heme. In its ferrous state, STEAP1 is capable of reducing transition metal ion complexes of Fe3+ and Cu2+. Ferrous STEAP1 also reacts readily with O2 through an outer sphere redox mechanism. Kinetics with all three substrates are biphasic with ∼80 and ∼20% for the fast and slow phases, respectively, in line with its heme heterogeneity. STEAP1 retained a low level of bound FAD during purification, and the binding equilibrium constant, KD, was ∼30 µM. These results highlight STEAP as a novel metal reductase and superoxide synthase and establish a solid basis for further research into understanding how STEAP1 activities may affect cancer progression.

H-NOX from Clostridium botulinum, like H-NOX from Thermoanaerobacter tengcongensis, Binds Oxygen but with a Less Stable Oxyferrous Heme Intermediate.

Heme nitric oxide/oxygen binding protein isolated from the obligate anaerobe Clostridium botulinum (Cb H-NOX) was previously reported to bind NO with a femtomolar K(D) (Nioche, P. et al. Science 2004, 306, 1550-1553). On the other hand, no oxyferrous Cb H-NOX was observed despite full conservation of the key residues that stabilize the oxyferrous complex in the H-NOX from Thermoanaerobacter tengcongensis (Tt H-NOX) (the same study). In this study, we re-measured the kinetics/affinities of Cb H-NOX for CO, NO, and O2. K(D)(CO) for the simple one-step equilibrium binding was 1.6 × 10(-7) M. The K(D)(NO) of Cb H-NOX was 8.0 × 10(-11) M for the first six-coordinate NO complex, and the previous femtomolar K(D)(NO) was actually an apparent K(D) for its multiple-step NO binding. An oxyferrous Cb H-NOX was clearly observed with a K(D)(O2) of 5.3 × 10(-5) M, which is significantly higher than Tt H-NOX's K(D)(O2) = 4.4 × 10(-8) M. The gaseous ligand binding of Cb H-NOX provides another supportive example for the "sliding scale rule" hypothesis (Tsai, A.-L. et al. Antioxid. Redox Signal. 2012, 17, 1246-1263), and the presence of hydrogen bond donor Tyr139 in Cb H-NOX selectively enhanced its affinity for oxygen.

Rational design of a fluorescent NADPH derivative imaging constitutive nitric-oxide synthases upon two-photon excitation.

We report the structure-based design and synthesis of a unique NOS inhibitor, called nanoshutter NS1, with two-photon absorption properties. NS1 targets the NADPH site of NOS by a nucleotide moiety mimicking NADPH linked to a conjugated push-pull chromophore with nonlinear absorption properties. Because NS1 could not provide reducing equivalents to the protein and competed with NADPH binding, it efficiently inhibited NOS catalysis. NS1 became fluorescent once bound to NOS with an excellent signal-to-noise ratio because of two-photon excitation avoiding interference from the flavin-autofluorescence and because free NS1 was not fluorescent in aqueous solutions. NS1 fluorescence enhancement was selective for constitutive NOS in vitro, in particular for endothelial NOS (eNOS). Molecular dynamics simulations suggested that two variable residues among NOS isoforms induced differences in binding of NS1 and in local solvation around NS1 nitro group, consistent with changes of NS1 fluorescence yield. NS1 colocalized with eNOS in living human umbilical vein endothelial cells. Thus, NS1 constitutes a unique class of eNOS probe with two-photon excitation in the 800-950-nm range, with great perspectives for eNOS imaging in living tissues.

Neurogranin modulates the Rate of Association between Calmodulin and Target Peptides.

The best-known mode of action of calmodulin (CaM) is binding of Ca 2+ to its N- and C-domains, followed by binding to target proteins. An underappreciated facet of this process is that CaM is typically bound to proteins at basal levels of free Ca 2+ , including the small, intrinsically disordered, neuronal IQ-motif proteins called PEP-19 and neurogranin (Ng). PEP-19 and Ng would not be effective competitive inhibitors of high-affinity Ca 2+ -dependent CaM targets at equilibrium since they bind to CaM with relatively low affinity, but they could influence the time course of CaM signaling by affecting the rate of association of CaM with high-affinity Ca 2+ -dependent targets. This mode of regulation may domain specific since PEP-19 binds to the C-domain of CaM, while Ng binds to both N- and C-domains. In this report, we used a model CaM binding peptide (CKIIp) to characterize the preferred pathway of complex formation with Ca 2+ -CaM at low levels of free Ca 2+ (0.25 to 1.5 µM), and how PEP-19 and Ng affect this process. We show that the dominant encounter complex involves association of CKIIp with the N-domain of CaM, even though the C-domain has a greater affinity for Ca 2+ . We also show that Ng greatly decreases the rate of association of Ca 2+ -CaM with CKIIp due to the relatively slow dissociation of Ng from CaM, and to interactions between the Gly-rich C-terminal region of Ng with the N-domain of CaM, which inhibits formation of the preferred encounter complex with CKIIp. These results provide the general mechanistic paradigms that binding CaM to targets can be driven by its N-domain, and that low-affinity regulators of CaM signaling have the potential to influence the rate of activation of high-affinity CaM targets and potentially affect the distribution of limited CaM among multiple targets during Ca 2+ oscillations. STATEMENT OF SIGNIFICANCE: Calmodulin is a small, essential regulator of multiple cellular processes including growth and differentiation. Its best-known mode of action is to first bind calcium and then bind and regulate the activity of target proteins. Each domain of CaM has distinct calcium binding properties and can interact with targets in distinct ways. We show here that the N-domain of calmodulin can drive its association with targets, and that a small, intrinsically disordered regulator of calmodulin signaling called neurogranin can greatly decrease the rate of association of CaM with high-affinity Ca 2+ -dependent targets. These results demonstrate the potential of neurogranin, and potentially other proteins, to modulate the time course of activation of targets by a limited intracellular supply of calmodulin.

Bi-directional allosteric pathway in NMDA receptor activation and modulation.

N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors involved in learning and memory. NMDA receptors primarily comprise two GluN1 and two GluN2 subunits. The GluN2 subunit dictates biophysical receptor properties, including the extent of receptor activation and desensitization. GluN2A- and GluN2D-containing receptors represent two functional extremes. To uncover the conformational basis of their functional divergence, we utilized single-molecule fluorescence resonance energy transfer to probe the extracellular domains of these receptor subtypes under resting and ligand-bound conditions. We find that the conformational profile of the GluN2 amino-terminal domain correlates with the disparate functions of GluN2A- and GluN2D-containing receptors. Changes at the pre-transmembrane segments inversely correlate with those observed at the amino-terminal domain, confirming direct allosteric communication between these domains. Additionally, binding of a positive allosteric modulator at the transmembrane domain shifts the conformational profile of the amino-terminal domain towards the active state, revealing a bidirectional allosteric pathway between extracellular and transmembrane domains.

The selectivity of Vibrio cholerae H-NOX for gaseous ligands follows the "sliding scale rule" hypothesis. Ligand interactions with both ferrous and ferric Vc H-NOX.

Vc H-NOX (or VCA0720) is an H-NOX (heme-nitric oxide and oxygen binding) protein from facultative aerobic bacterium Vibrio cholerae. It shares significant sequence homology with soluble guanylyl cyclase (sGC), a NO sensor protein commonly found in animals. Similar to sGC, Vc H-NOX binds strongly to NO and CO with affinities of 0.27 nM and 0.77 µM, respectively, but weakly to O2. When positioned on a "sliding scale" plot [Tsai, A.-l., et al. (2012) Biochemistry 51, 172-186], the line connecting log K(D)(NO) and log K(D)(CO) of Vc H-NOX can almost be superimposed with that of Ns H-NOX. Therefore, the measured affinities and kinetic parameters of gaseous ligands to Vc H-NOX provide more evidence to validate the "sliding scale rule" hypothesis. Like sGC, Vc H-NOX binds NO in multiple steps, forming first a six-coordinate heme-NO complex at a rate of 1.1 × 10(9) M(-1) s(-1), and then converts to a five-coordinate heme-NO complex at a rate that is also dependent on NO concentration. Although the formation of oxyferrous Vc H-NOX cannot be detected at a normal atmospheric oxygen level, ferrous Vc H-NOX is oxidized to the ferric form at a rate of 0.06 s(-1) when mixed with O2. Ferric Vc H-NOX exists as a mixture of high- and low-spin states and is influenced by binding to different ligands. Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lays the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX.

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site.

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.

A "sliding scale rule" for selectivity among NO, CO, and O2 by heme protein sensors.

Selectivity among NO, CO, and O2 is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (K(D)), the ratios for NO:CO:O2 binding stay roughly the same, 1:~10(3):~10(6), when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a "sliding scale rule" for plots of log(K(D)) versus ligand type that allows predictions of K(D) values if one or two are missing. The predicted K(D) for binding of O2to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O2 affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate, and imidazolate exert a "leveling" effect on ligand binding affinity. The reported picomolar K(D) for binding of NO to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO K(D) ratio of 1:~10(8). This deviation is explained by a complex, multistep process, in which an initial low-affinity hexacoordinate NO complex with a measured K(D) of ≈54 nM, matching that predicted from the sliding scale rule, is formed initially and then is converted to a high-affinity pentacoordinate complex. This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O2 binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation.

Dynamic ligand exchange in soluble guanylyl cyclase (sGC): implications for sGC regulation and desensitization.

Accumulating evidence indicates that the functional properties of soluble guanylyl cyclase (sGC) are affected not only by the binding of NO but also by the NO:sGC ratio and a number of cellular factors, including GTP. In this study, we monitored the time-resolved transformations of sGC and sGC-NO complexes generated with stoichiometric or excess NO in the presence and absence of GTP. We demonstrate that the initial five-coordinate sGC-NO complex is highly activated by stoichiometric NO but is unstable and transforms into a five-coordinate sGC-2 state. This sGC-2 rebinds NO to form a low activity sGC-NO complex. The stability of the initial complex is greatly enhanced by GTP binding, binding of an additional NO molecule, or substitution of ßHis-107. We propose that the transient nature of the sGC-NO complex, the formation of a desensitized sGC-2 state, and its transformation into a low activity sGC-NO adduct require ßHis-107. We conclude that conformational changes leading to sGC desensitization may be prevented by GTP binding to the catalytic site or by binding of an additional NO molecule to the proximal side of the heme. The implications of these observations for cellular NO/cGMP signaling and the process of rapid desensitization of sGC are discussed in the context of the proposed model of sGC/NO interactions and dynamic transformations.

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel.

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

Structural Arrangement Produced by Concanavalin A Binding to Homomeric GluK2 Receptors.

Kainate receptors are members of the ionotropic glutamate receptor family. They form cation-specific transmembrane channels upon binding glutamate that desensitize in the continued presence of agonists. Concanavalin A (Con-A), a lectin, stabilizes the active open-channel state of the kainate receptor and reduces the extent of desensitization. In this study, we used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the conformational changes underlying kainate receptor modulation by Con-A. These studies showed that Con-A binding to GluK2 homomeric kainate receptors resulted in closer proximity of the subunits at the dimer-dimer interface at the amino-terminal domain as well as between the subunits at the dimer interface at the agonist-binding domain. Additionally, the modulation of receptor functions by monovalent ions, which bind to the dimer interface at the agonist-binding domain, was not observed in the presence of Con-A. Based on these results, we conclude that Con-A modulation of kainate receptor function is mediated by a shift in the conformation of the kainate receptor toward a tightly packed extracellular domain.

Delta glutamate receptors are functional glycine- and á´ -serine-gated cation channels in situ.

Delta receptors are members of the ionotropic glutamate receptor superfamily and form trans-synaptic connections by interacting with the extracellular scaffolding protein cerebellin-1 and presynaptic transmembrane protein neurexin-1ß. Unlike other family members, however, direct agonist-gated ion channel activity has not been recorded in delta receptors. Here, we show that the GluD2 subtype of delta receptor forms cation-selective channels when bound to cerebellin-1 and neurexin-1ß. Using fluorescence lifetime measurements and chemical cross-linking, we reveal that tight packing of the amino-terminal domains of GluD2 permits glycine- and d-serine­induced channel openings. Thus, cerebellin-1 and neurexin-1ß act as biological cross-linkers to stabilize the extracellular domains of GluD2 receptors, allowing them to function as ionotropic excitatory neurotransmitter receptors in synapses.

A new paradigm for gaseous ligand selectivity of hemoproteins highlighted by soluble guanylate cyclase.

Nitric oxide (NO), carbon monoxide (CO), and oxygen (O2) are important physiological messengers whose concentrations vary in a remarkable range, [NO] typically from nM to several µM while [O2] reaching to hundreds of µM. One of the machineries evolved in living organisms for gas sensing is sensor hemoproteins whose conformational change upon gas binding triggers downstream response cascades. The recently proposed "sliding scale rule" hypothesis provides a general interpretation for gaseous ligand selectivity of hemoproteins, identifying five factors that govern gaseous ligand selectivity. Hemoproteins have intrinsic selectivity for the three gases due to a neutral proximal histidine ligand while proximal strain of heme and distal steric hindrance indiscriminately adjust the affinity of these three gases for heme. On the other hand, multiple-step NO binding and distal hydrogen bond donor(s) specifically enhance affinity for NO and O2, respectively. The "sliding scale rule" hypothesis provides clear interpretation for dramatic selectivity for NO over O2 in soluble guanylate cyclase (sGC) which is an important example of sensor hemoproteins and plays vital roles in a wide range of physiological functions. The "sliding scale rule" hypothesis has so far been validated by all experimental data and it may guide future designs for heme-based gas sensors.

Is Nostoc H-NOX a NO sensor or redox switch?

Nostoc sp. (Ns) H-NOX is a heme protein found in symbiotic cyanobacteria, which has approximately 35% sequence identity and high structural homology to the beta subunit of soluble guanylyl cyclase (sGC), suggesting a NO sensing function. However, UV-vis, EPR, NIR MCD, and ligand binding experiments with ferrous and ferric Ns H-NOX indicate significant functional differences between Ns H-NOX and sGC. (1) After NO binding to sGC, the proximal histidine dissociates from the heme iron, causing a conformational change that triggers activation of sGC. In contrast, formation of pentacoordinate (5c) NO heme occurs to only a limited extent in Ns H-NOX, even at >1 mM NO. (2) Unlike sGC, two different hexacoordinate (6c) NO complexes are formed in Ns H-NOX with initial and final absorbance peaks at 418 and 414 nm, and the conversion rate is linearly dependent on [NO], indicating that a second NO binds transiently to catalyze formation of the 414 nm species. (3) sGC is insensitive to oxygen, and ferric sGC prepared by ferricyanide oxidation has a 5c high-spin heme complex. In contrast, Ns H-NOX autoxidizes in 24 h if exposed to air and forms a 6c ferric heme complex, indicating a major conformational change after oxidation and coordination by a second histidine side chain. Such a large conformational transition suggests that Ns H-NOX could function as either a redox or a NO sensor in the cyanobacterium.

Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition.

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.