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 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 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.

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.

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.

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.

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.

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.

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.

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.

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.

The structural arrangement and dynamics of the heteromeric GluK2/GluK5 kainate receptor as determined by smFRET.

Kainate receptors, which are glutamate activated excitatory neurotransmitter receptors, predominantly exist as heteromers of GluK2 and GluK5 subunits in the mammalian central nervous system. There are currently no structures of the full-length heteromeric kainate receptors. Here, we have used single molecule FRET to determine the specific arrangement of the GluK2 and GluK5 subunits within the dimer of dimers configuration in a full-length receptor. Additionally, we have also studied the dynamics and conformational heterogeneity of the amino-terminal and agonist-binding domain interfaces associated with the resting and desensitized states of the full-length heteromeric kainate receptor using FRET-based methods. The smFRET data are compared to similar experiments performed on the homomeric kainate receptor to provide insight into the differences in conformational dynamics that distinguish the two functionally. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Mechanism of modulation of AMPA receptors by TARP-γ8.

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

Critical Comparison of the Superoxide Dismutase-like Activity of Carbon Antioxidant Nanozymes by Direct Superoxide Consumption Kinetic Measurements.

The superoxide dismutase-like activity of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs), anthracite and bituminous graphene quantum dots (PEG-aGQDs and PEG-bGQDs, respectively), and two fullerene carbon nanozymes, tris malonyl-C60 fullerene (C3) and polyhydroxylated-C60 fullerene (C60-OHn), were compared using direct optical stopped-flow kinetic measurements, together with three native superoxide dismutases (SODs), CuZnSOD, MnSOD, and FeSOD, at both pH 12.7 and 8.5. Computer modeling including both SOD catalytic steps and superoxide self-dismutation enabled the best choice of catalyst concentration with minimal contribution to the observed kinetic change from the substrate self-dismutation. Biexponential fitting to the kinetic data ranks the rate constant (M-1 s-1) in the order of PEG-HCCs > CuZnSOD ≈ MnSOD ≈ PEG-aGQDs ≈ PEG-bGQDs > FeSOD ≫ C3 > C60-OHn at pH 12.7 and MnSOD > CuZnSOD ≈ PEG-HCCs > FeSOD > PEG-aGQDs ≈ PEG-bGQDs ≫ C3 ≈ C60-OHn at pH 8.5. Nonlinear regression of the kinetic model above yielded the same ranking as the biexponential fit, but provided better mechanistic insight. The data obtained by freeze-quench EPR direct assay at pH 12.7 also yield the same ranking as stopped-flow data. This is a necessary assessment of a panel of proclaimed carbon nano SOD mimetics using the same two direct methods, revealing a dramatic, 3-4 orders of magnitude difference in SOD activity between PEG-HCCs/PEG-GQDs from soluble fullerenes.

Catalytic oxidation and reduction reactions of hydrophilic carbon clusters with NADH and cytochrome C: features of an electron transport nanozyme.

Previously, our group reported on the promising efficacy of poly(ethylene glycol)-hydrophilic carbon clusters (PEG-HCCs) to work as broadly active and high capacity antioxidants in brain ischemia and injury models including stroke and traumatic brain injury coupled with hemorrhagic shock. PEG-HCCs are a carbon nanomaterial derived from harsh oxidation of single wall carbon nanotubes and covalently modified with poly(ethylene glycol). They retain no tubular remnants and are composed of a highly oxidized carbon core functionalized with epoxy, peroxyl, quinone, ketone, carboxylate, and hydroxyl groups. HCCs are the redox active carbon core of PEG-HCCs, which have a broad reduction potential range starting at +200 mV and extending to -2 V. Here we describe a new property of these materials: the ability to catalytically transfer electrons between key surrogates and proteins of the mitochondrial electron transport complex in a catalytic fashion consistent with the concept of a nanozyme. The estimated reduction potential of PEG-HCCs is similar to that of ubiquinone and they enabled the catalytic transfer of electrons from low reduction potential species to higher reduction electron transport complex constituents. PEG-HCCs accelerated the reduction of resazurin (a test indicator of mitochondrial viability) and cytochrome c by NADH and ascorbic acid in solution. Kinetic experiments suggested a transient tertiary complex. Electron paramagnetic resonance demonstrated NADH increased the magnitude of PEG-HCCs' intrinsic radical, which then reduced upon subsequent addition of cytochrome c or resazurin. Deconvolution microscopy identified PEG-HCCs in close proximity to mitochondria after brief incubation with cultured SHSY-5Y human neuroblastoma cells. Compared to methylene blue (MB), considered a prototypical small molecule electron transport shuttle, PEG-HCCs were more protective against toxic effects of hydrogen peroxide in vitro and did not demonstrate impaired cell viability as did MB. PEG-HCCs were protective in vitro when cells were exposed to sodium cyanide, a mitochondrial complex IV poison. Because mitochondria are a major source of free radicals in pathology, we suggest that this newly described nanozyme action helps explain their in vivo efficacy in a range of injury models. These findings may also extend their use to mitochondrial disorders.

The structural arrangement at intersubunit interfaces in homomeric kainate receptors.

Kainate receptors are glutamate-gated cation-selective channels involved in excitatory synaptic signaling and are known to be modulated by ions. Prior functional and structural studies suggest that the dimer interface at the agonist-binding domain plays a key role in activation, desensitization, and ion modulation in kainate receptors. Here we have used fluorescence-based methods to investigate the changes and conformational heterogeneity at these interfaces associated with the resting, antagonist-bound, active, desensitized, and ion-modulated states of the receptor. These studies show that in the presence of Na+ ions the interfaces exist primarily in the coupled state in the apo, antagonist-bound and activated (open channel) states. Under desensitizing conditions, the largely decoupled dimer interface at the agonist-binding domain as seen in the cryo-EM structure is one of the states observed. However, in addition to this state there are several additional states with lower levels of decoupling. Replacing Na+ with Cs+ does not alter the FRET efficiencies of the states significantly, but shifts the population to the more decoupled states in both resting and desensitized states, which can be correlated with the lower activation seen in the presence of Cs+.

Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants.

Graphene quantum dots (GQDs) have recently been employed in various fields including medicine as antioxidants, primarily because of favorable biocompatibility in comparison to common inorganic quantum dots, although the structural features that lead to the biological activities of GQDs are poorly understood. Here, we report that coal-derived GQDs and their poly(ethylene glycol)-functionalized derivatives serve as efficient antioxidants, and we evaluate their electrochemical, chemical, and in vitro biological activities.

Efficacy of Novel Carbon Nanoparticle Antioxidant Therapy in a Severe Model of Reversible Middle Cerebral Artery Stroke in Acutely Hyperglycemic Rats.

INTRODUCTION: While oxidative stress can be measured during transient cerebral ischemia, antioxidant therapies for ischemic stroke have been clinically unsuccessful. Many antioxidants are limited in their range and/or capacity for quenching radicals and can generate toxic intermediates overwhelming depleted endogenous protection. We developed a new antioxidant class, 40 nm × 2 nm carbon nanoparticles, hydrophilic carbon clusters, conjugated to poly(ethylene glycol) termed PEG-HCCs. These particles are high-capacity superoxide dismutase mimics, are effective against hydroxyl radical, and restore the balance between nitric oxide and superoxide in the vasculature. Here, we report the effects of PEG-HCCs administered during reperfusion after transient middle cerebral artery occlusion (tMCAO) by suture in the rat under hyperglycemic conditions. Hyperglycemia occurs in one-third of stroke patients and worsens clinical outcome. In animal models, this worsening occurs largely by accelerating elaboration of reactive oxygen species (ROS) during reperfusion. METHODS: PEG-HCCs were studied for their protective ability against hydrogen peroxide in b.End3 brain endothelial cell line and E17 primary cortical neuron cultures. In vivo, hyperglycemia was induced by streptozotocin injection 2 days before tMCAO. 58 Male Sprague-Dawley rats were analyzed. They were injected IV with PBS or PEG-HCCs (4 mg/kg 2×) at the time of recanalization after either 90- or 120-min occlusion. Rats were survived for up to 3 days, and infarct volume characteristics and neurological functional outcome (modified Bederson Score) were assessed. RESULTS: PEG-HCCs were protective against hydrogen peroxide in both culture models. In vivo improvement was found after PEG-HCCs with 90-min ischemia with reduction in infarct size (42%), hemisphere swelling (46%), hemorrhage score (53%), and improvement in Bederson score (70%) (p = 0.068-0.001). Early high mortality in the 2-h in the PBS control group precluded detailed analysis, but a trend was found in improvement in all factors, e.g., reduction in infarct volume (48%; p = 0.034) and a 56% improvement in Bederson score (p = 0.055) with PEG-HCCs. CONCLUSION: This nano-antioxidant showed some improvement in several outcome measures in a severe model of tMCAO when administered at a clinically relevant time point. Long-term studies and additional models are required to assess potential for clinical use, especially for patients hyperglycemic at the time of their stroke, as these patients have the worst outcomes.

Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease.

Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.

Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase.

To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O2, including KD, kon, and koff, of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The KD(NO) and KD(CO) of each bacterial H-NOX or sGC follow the "sliding scale rule"; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold.

Perylene Diimide as a Precise Graphene-like Superoxide Dismutase Mimetic.

Here we show that the active portion of a graphitic nanoparticle can be mimicked by a perylene diimide (PDI) to explain the otherwise elusive biological and electrocatalytic activity of the nanoparticle construct. Development of molecular analogues that mimic the antioxidant properties of oxidized graphenes, in this case the poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs), will afford important insights into the highly efficient activity of PEG-HCCs and their graphitic analogues. PEGylated perylene diimides (PEGn-PDI) serve as well-defined molecular analogues of PEG-HCCs and oxidized graphenes in general, and their antioxidant and superoxide dismutase-like (SOD-like) properties were studied. PEGn-PDIs have two reversible reduction peaks, which are more positive than the oxidation peak of superoxide (O2•-). This is similar to the reduction peak of the HCCs. Thus, as with PEG-HCCs, PEGn-PDIs are also strong single-electron oxidants of O2•-. Furthermore, reduced PEGn-PDI, PEGn-PDI•-, in the presence of protons, was shown to reduce O2•- to H2O2 to complete the catalytic cycle in this SOD analogue. The kinetics of the conversion of O2•- to O2 and H2O2 by PEG8-PDI was measured using freeze-trap EPR experiments to provide a turnover number of 133 s-1; the similarity in kinetics further supports that PEG8-PDI is a true SOD mimetic. Finally, PDIs can be used as catalysts in the electrochemical oxygen reduction reaction in water, which proceeds by a two-electron process with the production of H2O2, mimicking graphene oxide nanoparticles that are otherwise difficult to study spectroscopically.