The synthesis, structure, and spectroscopic signatures of a series of four-coordinate iron(II) complexes of β-ketoiminates and their zinc(II) analogues are presented. An unusual five-coordinate iron(II) triflate with three oxygen bound protonated β-ketoimines is also synthesized and structurally characterized. Single-crystal X-ray crystallographic analysis reveals that the deprotonated bis(chelate)metal complexes are four-coordinate with various degrees of distortion depending on the degree of steric bulk and the electronics of the metal center. Each of the high-spin iron(II) centers exhibits multiple electronic transitions including ligand π to π*, metal-to-ligand charge transfer, and spin-forbidden d-d bands. The (1)H NMR spectra of the paramagnetic high-spin iron(II) centers are assigned on the basis of chemical shifts, longitudinal relaxation times (T(1)), relative integrations, and substitution of the ligands. The electrochemical studies support variations in the ligand strength. Parallel mode EPR measurements for the isopropyl substituted ligand complex of iron(II) show low-field resonances (g > 9.5) indicative of complex aggregation or crystallite formation. No suitable solvent system or glassing mixture was found to remedy this phenomenon. However, the bulkier diisopropylphenyl substituted ligand exhibits an integer spin signal consistent with an isolated iron(ii) center [S = 2; D = -7.1 ± 0.8 cm(-1); E/D = 0.1]. A tentative molecular orbital diagram is assembled.

Solution pH and the pKa values of ionizable residues are critical factors known to influence enzyme catalysis, structural stability, and dynamical fluctuations. Presented here is an exhaustive computational study utilizing long time constant pH molecular dynamics, pH replica exchange simulations, and kinetic modeling to evaluate pH-dependent conformations, charge dynamics, residue pKa values, and the catalytic activity-pH profile for cellobiohydrolase Cel7B from Melanocarpus albomyces . The predicted pKa values support the role of Glu212 as the catalytic nucleophile and Glu217 as the acid-base residue. The presence of a charge-correlated active site and an extensive hydrogen bonding network is found to be critical in enabling favorable residue orientations for catalysis and shuttling excess protons around the active site. Clusters of amino acids are identified that act in concert to effectively modulate the optimal pH for catalysis while elevating the overall catalytic rate with respect to a noncoupled system. The work presented here demonstrates the complex and critical role of coupled ionizable residues to the proper functioning of cellobiohydrolase Cel7B, functionally related glycosyl hydrolases, and enzymes in general. The simulations also support the use of the CpHMD for the accurate prediction of residue pKa values and to evaluate the impact of pH on protein structure and charge dynamics.

α-Conotoxin MII (α-CTxMII) is a potent and selective peptide antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). Studies have shown that His9 and His12 are significant determinants of toxin binding affinity for nAChR, while Glu11 may dictate differential toxin affinity between nAChR isoforms. The protonation state of these histidine residues and therefore the charge on the α-conotoxin may contribute to the observed differences in binding affinity and selectivity. In this study, we assess the pH dependence of the protonation state of His9 and His12 by 1H NMR spectroscopy and constant pH molecular dynamics (CpHMD) in α-CTxMII, α-CTxMII[E11A], and the triple mutant, α-CTxMII[N5R:E11A:H12K]. The E11A mutation does not significantly perturb the pKa of His9 or His12, while N5R:E11A:H12K results in a significant decrease in the pKa value of His9. The pKa values predicted by CpHMD simulations are in good agreement with 1H NMR spectroscopy, with a mean absolute deviation from experiment of 0.3 pKa units. These results support the use of CpHMD as an efficient and inexpensive predictive tool to determine pKa values and structural features of small peptides critical to their function.

The size and character of the peripheral loops enclosing the active site for cellulase enzymes is believed to play a major role in dictating many critical enzymatic properties. For many cellulases it is observed that fully enclosed active sites forming a tunnel are more conducive to cellobiohydrolase activity and the ability to processively move along the substrate. Conversely, a more open active site groove is indicative of endoglucanase activity. For both cellobiohydrolases and endoglucanases, the loop regions have been implicated in the ability of the enzyme to bind substrate, influence the pKa of active site residues, modulate the catalytic activity, and influence thermal stability. Reported here are constant pH molecular dynamics (CpHMD) simulations that investigate the role of dynamic fluctuations, substrate interactions, and residue pKa values for the peripheral loops enclosing the active site of the cellobiohydrolase Melanocarpus albomyces Cel7B. Two highly flexible loop regions in the free enzyme have been identified, which impact the overall dynamical motions of the enzyme. Charge interactions between Asp198 and Asp367, which reside on two adjacent loops, were found to influence the overall loop conformations and dynamics. In the presence of a substrate the protonation of Asp367, Asp198, and Tyr370 were found to stabilize substrate binding and control the movement of two peripheral loops onto the active site containing the substrate (i.e., clamping down). The substrate-induced response of the loop regions secures the cellulose polymer in the catalytic tunnel and creates an environment that is conducive to hydrolysis of the glycosidic bond.

The ability to utilize biomass as a feedstock for liquid fuel and value-added chemicals is dependent on the efficient and economic utilization of lignin, hemicellulose, and cellulose. In current bioreactors, cellulases are used to convert crystalline and amorphous cellulose to smaller oligomers and eventually glucose by means of cellulase enzymes. A critical component of the enzyme catalyzed hydrolysis reaction is the degree to which the enzyme can facilitate substrate ring deformation from the chair to a more catalytically active conformation (e.g. skewed boat) at the -1 subsite. Presented here is an evaluation of the impact of the protonation state for critical active site residues (i.e. Glu212, Asp214, Glu217, and His228) in Melanocarpus albomyces (Ma) Cellobiohydrolase Cel7B on the substrate's orientation and ring conformation. It is found that the protonation state of the active site can disrupt the intra-enzyme hydrogen bonding network and enhance the sampling of various ring puckering conformations for the substrate ring at the +1 and -1 subsites. In particular it is observed that the protonation state of Asp214 dictates the accessibility of the glycosidic bond to the catalytic acid/base Glu217 by influencing the φ/ψ dihedral angles and the puckering of the ring structure. The protonation-orientation-conformation analysis has revealed an active site that primarily utilizes two highly coupled protonation schemes; one protonation scheme to orient the substrate and generate catalytically favorable substrate geometries and ring puckering conformations and another protonation scheme to hydrolyze the glycosidic bond. In addition to identifying how enzymes utilize protonation state to manipulate substrate geometry, this study identifies possible directions for improving catalytic activity through protein engineering.

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