Ionizable Residues Research Articles

Improved Process Stability on an Extremely Thin Amorphous/Crystalline Silicon Interface Passivation Layer by Using Predeposition on the Chamber Wall

In this study, in situ plasma diagnostic systems of optical emission spectrometry (OES) and quadrupole mass spectrometry (QMS) were used to monitor an extremely thin (5–10 nm) intrinsic amorphous/crystalline Si interface passivation film as deposited using plasma-enhanced chemical vapor deposition (PECVD). We observed a dramatic improvement in the quality of the passivation layer with a chamber background environment, even with a background pressure of 1E-6 Torr. When the chamber walls were coated (at a predeposition time of approximately 150 min) to a specified thickness of a few hundred micrometers, the minority carrier lifetime increased by more than 26 times, as compared with an insufficiently coated counterpart with a predeposition of approximately 30 min (from approximately 30 μs to 800 μs). In this predeposition process, the tendency of species to concentrate could be systematically obtained using the in situ OES system, with the chamber environment monitored using residual gas analysis and threshold ionization mass spectrometry of QMS. We found that the optimal predeposition time was 150 min, which enabled the OES intensity of Si*/SiH*, Hβ/Hα, and Hα/SiH* to become stable. OES and QMS were incorporated in this study and were validated using the Fourier-transform infrared spectroscopy absorbance spectra on the films. The plasma condition was stabilized and the minority carrier lifetime was improved to 800 μs. The proposed predeposition process stabilized the chamber environment and gas discharge. Therefore, the optimized value of the minority carrier lifetime could be consistently obtained. A transmission electron microscopy photograph showed a compact a-Si:H layer (of approximately 10 nm) on the c-Si substrate interface passivation layer with a void-free and crystallites-free interface after a predeposition time of 150 min.

Effects of pH and Salt Concentration on Stability of a Protein G Variant Using Coarse-Grained Models

The importance of charge-charge interactions in the thermal stability of proteins is widely known. pH and ionic strength play a crucial role in these electrostatic interactions, as well as in the arrangement of ionizable residues in each protein-folding stage. In this study, two coarse-grained models were used to evaluate the effect of pH and salt concentration on the thermal stability of a protein G variant (1PGB-QDD), which was chosen due to the quantity of experimental data exploring these effects on its stability. One of these coarse-grained models, the TKSA, calculates the electrostatic free energy of the protein in the native state via the Tanford-Kirkwood approach for each residue. The other one, CpHMD-SBM, uses a Coulomb screening potential in addition to the structure-based model Cα. Both models simulate the system in constant pH. The comparison between the experimental stability analysis and the computational results obtained by these simple models showed a good agreement. Through the TKSA method, the role of each charged residue in the protein’s thermal stability was inferred. Using CpHMD-SBM, it was possible to evaluate salt and pH effects throughout the folding process. Finally, the computational pKa values were calculated by both methods and presented a good level of agreement with the experiments. This study provides, to our knowledge, new information and a comprehensive description of the electrostatic contribution to protein G stability.

Design of a software for calculating isoelectric point of a polypeptide according to their net charge using the graphical programming language LabVIEW.

A software to calculate the net charge and to predict the isoelectric point (pI) of a polypeptide is developed in this work using the graphical programming language LabVIEW. Through this instrument the net charges of the ionizable residues of the polypeptide chains of the proteins are calculated at different pH values, tabulated, pI is predicted and an Excel (-xls) type file is generated. In this work, the experimental values of the pIs (pI) of different proteins are compared with the values of the pIs (pI) calculated graphically, achieving a correlation coefficient (R) of 0.934746 which represents a good reliability for a p < 0.01. In this way the generated program can constitute an instrument applicable in the laboratory, facilitating the calculation to graduate students and junior researchers. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):39-46, 2018.

Proton Network Flexibility Enables Robustness and Large Electric Fields in the Ketosteroid Isomerase Active Site.

Hydrogen-bond networks play vital roles in biological functions ranging from protein folding to enzyme catalysis. Here we combine electronic structure calculations and ab initio path integral molecular dynamics simulations, which incorporate both nuclear and electronic quantum effects, to show why the network of short hydrogen bonds in the active site of ketosteroid isomerase is remarkably robust to mutations along the network and how this gives rise to large local electric fields. We demonstrate that these properties arise from the network's ability to respond to a perturbation by shifting proton positions and redistributing electronic charge density. This flexibility leads to small changes in properties such as the partial ionization of residues and pKa isotope effects upon mutation of the residues, consistent with recent experiments. This proton flexibility is further enhanced when an extended hydrogen-bond network forms in the presence of an intermediate analogue, which allows us to explain the chemical origins of the large electric fields in the enzyme's active site observed in recent experiments.

PH Dependence of Charge Multipole Moments in Proteins

Electrostatic interactions play a fundamental role in the structure and function of proteins. Due to ionizable amino acid residues present on the solvent-exposed surfaces of proteins, the protein charge is not constant but varies with the changes in the environment—most notably, the pH of the surrounding solution. We study the effects of pH on the charge of four globular proteins by expanding their surface charge distributions in terms of multipoles. The detailed representation of the charges on the proteins is in this way replaced by the magnitudes and orientations of the multipole moments of varying order. Focusing on the three lowest-order multipoles—the total charge, dipole, and quadrupole moment—we show that the value of pH influences not only their magnitudes, but more notably and importantly also the spatial orientation of their principal axes. Our findings imply important consequences for the study of protein-protein interactions and the assembly of both proteinaceous shells and patchy colloids with dissociable charge groups.

A comprehensive analysis of the computed tautomer fractions of the imidazole ring of histidines in Loligo vulgaris

A recently introduced electrostatic-based method to determine the pKa values of ionizable residues and fractions of ionized and tautomeric forms of histidine (His) and acid residues in proteins, at a given fixed pH, is applied here to the analysis of a His-rich protein, namely Loligo vulgaris (pdb id 1E1A), a 314-residue all-β protein. The average tautomeric fractions for the imidazole ring of each of the six histidines in the sequence were computed using an approach that includes, but is not limited to, molecular dynamic simulations coupled with calculations of the ionization states for all 94 ionizable residues of protein 1E1A in water at pH 6.5 and 300 K. The electrostatic-calculated tautomeric fractions of the imidazole ring of His were compared with predictions obtained from an existent NMR-based methodology. Our results indicate that: (i) the averaged electrostatic-based tautomeric predictions for the imidazole ring of all histidines of Loligo vulgaris are dominated by the Nε2-H rather than the Nδ1-H form, although such preferences from the NMR-based methodology are not so well defined; (ii) the computed average absolute difference between the electrostatic- and the NMR-based tautomeric predictions among all six histidines vary among 0% to 17%; (iii) for the His showing the largest fraction of the neutral form (81%), the absolute difference between the NMR- and electrostatic-based computed tautomeric predictions is only 3%; and (iv) the tautomeric predictions for the imidazole ring of His computed with the NMR-based methodology are stable within a certain, well-defined, range of variations of a tautomer-related parameter.

Protonation States of Homocitrate and Nearby Residues in Nitrogenase Studied by Computational Methods and Quantum Refinement.

Nitrogenase is the only enzyme that can break the triple bond in N2 to form two molecules of ammonia. The enzyme has been thoroughly studied with both experimental and computational methods, but there is still no consensus regarding the atomic details of the reaction mechanism. In the most common form, the active site is a MoFe7S9C(homocitrate) cluster. The homocitrate ligand contains one alcohol and three carboxylate groups. In water solution, the triply deprotonated form dominates, but because the alcohol (and one of the carboxylate groups) coordinate to the Mo ion, this may change in the enzyme. We have performed a series of computational calculations with molecular dynamics (MD), quantum mechanical (QM) cluster, combined QM and molecular mechanics (QM/MM), QM/MM with Poisson-Boltzmann and surface area solvation, QM/MM thermodynamic cycle perturbations, and quantum refinement methods to settle the most probable protonation state of the homocitrate ligand in nitrogenase. The results quite conclusively point out a triply deprotonated form (net charge -3) with a proton shared between the alcohol and one of the carboxylate groups as the most stable at pH 7. Moreover, we have studied eight ionizable protein residues close to the active site with MD simulations and determined the most likely protonation states.

Separation of cellulose nanowhiskers from microcrystalline cellulose with an aqueous protic ionic liquid based on ammonium and hydrogensulphate

Cellulose nanowhiskers (NWC) have several applications, but its achievement, when held by hydrolysis with strong acids, presents some undesirable aspects as their corrosivity, toxicity and low recovery rate. Recently, [BMIM][HSO4] was proposed as an alternative, but its use has yet disadvantages, mainly due its toxicity and cost. In this sense, this work reports the separation of NWC from MCC by a synthetized protic ionic liquid 2-hydroxyethylamonium hydrogensulphate ([2-HEA][HSO4]) potentially lower toxic than [BMIM][HSO4], which can be easily produced by a low-cost procedure. A stable and polydisperse NWC aqueous suspension was formed exhibiting the birefringence flow typical of nanoparticle dispersions. TEM micrographies confirm needle-shape particles with high average aspect ratio of 50.23 ± 20.93 nm. A good thermal stability was achieved, with Tmax of cellulose decomposition around 234 °C. No ionic liquid residues were observed in the NWC by FTIR analysis. The calculated crystallinity index and crystallite size were 54% and 17.2 nm, respectively. The ionic liquid used in the hydrolysis experiments was successfully recovered in 76.91 ± 0.13% yield, with a similar structure to that of the original ionic liquid. Therefore, [2-HEA][HSO4] was successfully tested as a new, alternative and promising medium to separate cellulose nanowhiskers from cellulosic sources.

Electrohydrodynamic patterning in a curable resin over a wide range of fabrication parameters

This work reports the first use of a room-temperature curing epoxy resin to produce microscopic arrays of ordered 2-D structures via electrohydrodynamic instability (EHD) patterning. The measured spacing of these structures, and the formation of novel ‘starburst’ shapes, support a leaky dielectric theoretical model over perfect dielectric ones. The existence of the starbursts implies that traces of ionic residues in the uncured epoxy should be taken into account in any model of the patterning process, and their formation implies that mobile surface charges arising from trace ionic impurities must be considered when seeking to achieve smaller length-scale structures in epoxy by EHD using flat electrodes. Samples patterned in the μm range showed mid-infrared spectral features with wavelength maxima that broadly scaled with the pillar spacings in the films.

Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions

The assembly of integral membrane protein complexes is frequently supported by transmembrane domain (TMD) interactions. Here, we present the BLaTM assay that measures homotypic as well as heterotypic TMD-TMD interactions in a bacterial membrane. The system is based on complementation of β-lactamase fragments genetically fused to interacting TMDs, which confers ampicillin resistance to expressing cells. We validated BLaTM by showing that the assay faithfully reports known sequence-specific interactions of both types. In a practical application, we used BLaTM to screen a focussed combinatorial library for heterotypic interactions driven by electrostatic forces. The results reveal novel patterns of ionizable amino acids within the isolated TMD pairs. Those patterns indicate that formation of heterotypic TMD pairs is most efficiently supported by closely spaced ionizable residues of opposite charge. In addition, TMD heteromerization can apparently be driven by hydrogen bonding between basic or between acidic residues.

Mechanism of formate-nitrite transporters by dielectric shift of substrate acidity.

Bacterial formate-nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate-nitrite transporters.

Coupled molecular dynamics and continuum electrostatic method to compute the ionization pKa’s of proteins as a function of pH. Test on a large set of proteins

A computational method, to predict the pKa values of the ionizable residues Asp, Glu, His, Tyr, and Lys of proteins, is presented here. Calculation of the electrostatic free-energy of the proteins is based on an efficient version of a continuum dielectric electrostatic model. The conformational flexibility of the protein is taken into account by carrying out molecular dynamics simulations of 10 ns in implicit water. The accuracy of the proposed method of calculation of pKa values is estimated from a test set of experimental pKa data for 297 ionizable residues from 34 proteins. The pKa-prediction test shows that, on average, 57, 86, and 95% of all predictions have an error lower than 0.5, 1.0, and 1.5 pKa units, respectively. This work contributes to our general understanding of the importance of protein flexibility for an accurate computation of pKa, providing critical insight about the significance of the multiple neutral states of acid and histidine residues for pKa-prediction, and may spur significant progress in our effort to develop a fast and accurate electrostatic-based method for pKa-predictions of proteins as a function of pH.

Proteins as pH Sensors and Switches

Ionizable residues buried in dry or hydrophobic environments in proteins can have extremely anomalous pKa values (pKa > 10 for Asp and Glu, pKa 5 for Lys). These buried ionizable groups are involved in many forms of biological energy transduction. Their anomalous pKa values are usually essential for function in many pH-driven processes. Anomalous pKa values impart unusual pH sensitivity to the stability of the folded state. Previously we examined energetic consequences of burial of Lys, His, Arg, Asp and Glu in hydrophobic environments systematically, using staphylococcal nuclease (SNase) as a model system. With NMR spectroscopy and X-ray crystallography we have now characterized a variety of different types of conformational reorganization coupled to the ionization of these buried groups. Not only are conformational transitions driven by the binding or release of an H+ the basis of many H+-coupled energy transduction processes (e.g. ATPase), the detailed structural characterization of locally, partially or subglobally unfolded forms of SNase has also contributed a unique perspective on its folding landscape. When they occur, these structural transitions determine the magnitude of the shift in the pKa of internal groups. We have now also demonstrated that the unusual properties of buried ionizable groups can be harnessed to engineer proteins capable of acting as pH sensors and pH-driven switches. These switch proteins are sensitive to changes in pH in the physiological range despite using Asp, Glu or Lys residues for purposes of pH sensing. I will describe specific examples illustrating general and transferable engineering principles that may be used to turn any protein into a pH-sensitive protein with structure or function that can be modulated by small changes in pH in the physiological range.

Solid-State NMR Investigations of Transmembrane Helix Interactions

Transmembrane proteins are vital to numerous biological processes, including signal transduction and ion conduction. In many cases, the behavior of potentially ionizable residues in the hydrophobic interior of the membrane is crucial to understanding protein function. We have employed a low-dynamic model α-helical transmembrane peptide, GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide) as a framework to investigate the possibility of (+/-) ion-pair interactions between arginine and glutamic acid side chains located on different and independent (separately diffusing) transmembrane helices, by 2H NMR spectroscopy. One peptide with an Arg residue, GWALP23-R14 (acetyl-GGALWLALALALARALALWLAGA-amide), and another with a Glu residue, GWALP23-E14 (acetyl-GGALWLALALALAEALALWLAGA-amide), were incorporated into DOPC bilayers together (1:67 peptide:lipid ratio). Past experiments have shown that GWALP23-R14 alone is well-aligned in DOPC bilayers, gives narrow resonances for 2H-labeled alanines and remains charged, even under alkaline pH conditions (JACS 2010, 132(16):5803). By contrast, 2H-Ala-labeled GWALP23-E14 exhibits much broader 2H NMR spectra. We now find that the spectra of labeled GWALP23-R14 become noticeably broadened and weakened, without changing the quadrupolar splittings, when even 16 mol% GWALP23-E14 is included with GWALP23-R14 in the DOPC bilayer samples. A control peptide, GWALP23, has little effect on GWALP23-R14 at 16 mol%, but spectral broadening and weakening occur with at 50 mol%. Conversely, the addition of GWALP-R14 (16 or 50 mol%), has no effect on the already broad spectra of labeled GWALP-E14 in DOPC bilayers. Preliminary results, therefore, suggest that while GWALP-E14 causes concentration-dependent effects on the spectra of GWALP-R14, the complex or possibly multi-state behavior of GWALP-E14 is not ‘rescued’ by the addition of GWALP-R14 in DOPC bilayers.

Transition from disordered aggregates to ordered lattices: kinetic control of the assembly of a computationally designed peptide.

Natural biomolecular self-assembly typically occurs under a narrow range of solution conditions, and the design of sequences that can form prescribed structures under a range of such conditions would be valuable in the bottom-up assembly of predetermined nanostructures. We present a computationally designed peptide that robustly self-assembles into regular arrays under a wide range of solution pH and temperature conditions. Controling the solution conditions provides the opportunity to exploit a simple and reproducible approach for altering the pathway of peptide solution self-assembly. The computationally designed peptide forms a homotetrameric coiled-coil bundle that further self-assembles into 2-D plate structures with well-defined inter-bundle symmetry. Herein, we present how modulation of solution conditions, such as pH and temperature, can be used to control the kinetics of the inter-bundle assembly and manipulate the final morphology. Changes in solution pH primarily influence the inter-bundle assembly by affecting the charged state of ionizable residues on the bundle exterior while leaving the homotetrameric coiled-coil structure intact. At low pH, repulsive interactions prevent 2-D lattice nanostructure formation. Near the estimated isoelectric point of the peptide, bundle aggregation is rapid and yields disordered products, which subsequently transform into ordered nanostructures over days to weeks. At elevated temperatures (T = 40 °C or 50 °C), the formation of disordered, kinetically-trapped products largely can be eliminated, allowing the system to quickly assemble into plate-like nanostructured lattices. Moreover, subtle changes in pH and in the peptide charge state have a significant influence on the thickness of formed plates and on the hierarchical manner in which plates fuse into larger material structures with observable grain boundaries. These findings confirm the ability to finely tune the peptide assembly process to achieve a range of engineered structures with one simple 29-residue peptide building block.

Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism.

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5–6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a ‘molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.

Active Site Desolvation and Thermostability Trade-Offs in the Evolution of Catalytically Diverse Triazine Hydrolases

The desolvation of ionizable residues in the active sites of enzymes and the subsequent effects on catalysis and thermostability have been studied in model systems, yet little about how enzymes can naturally evolve to include active sites with highly reactive and desolvated charges is known. Variants of triazine hydrolase (TrzN) with significant differences in their active sites have been isolated from different bacterial strains: TrzN from Nocardioides sp. strain MTD22 contains a catalytic glutamate residue (Glu241) that is surrounded by hydrophobic and aromatic second-shell residues (Pro214 and Tyr215), whereas TrzN from Nocardioides sp. strain AN3 has a noncatalytic glutamine residue (Gln241) at an equivalent position, surrounded by hydrophilic residues (Thr214 and His215). To understand how and why these variants have evolved, a series of TrzN mutants were generated and characterized. These results show that desolvation by second-shell residues increases the pKa of Glu241, allowing it to act as a general acid at neutral pH. However, significant thermostability trade-offs are required to incorporate the ionizable Glu241 in the active site and to then enclose it in a hydrophobic microenvironment. Analysis of high-resolution crystal structures shows that there are almost no structural changes to the overall configuration of the active site due to these mutations, suggesting that the changes in activity and thermostability are purely based on the altered electrostatics. The natural evolution of these enzyme isoforms provides a unique system in which to study the fundamental process of charged residue desolvation in enzyme catalysis and its relative contribution to the creation and evolution of an enzyme active site.

Structural Stability, Transitions, and Interactions within SoxYZCD-Thiosulphate from Sulfurimonas denitrificans: An In Silico Molecular Outlook for Maintaining Environmental Sulphur Cycle.

Thiosulphate oxidation (an essential mechanism) serves to maintain the global sulphur cycle. Earlier experimental and computational studies dealt with environmental thiosulphate oxidation but none dealt with thiosulphate oxidation from deep ocean belts. Wet-laboratory experimental research shows that epsilon-proteobacteria Sulfurimonas denitrificans possess sox (sulphur-oxidizing) operon and perform thiosulphate oxidation efficiently underneath the oceans. From this specific sox operon, SoxCD complex recycles the thiosulphate-bound SoxY from SoxYZ complex to balance the environmental sulphur cycle. So, four chief proteins were variedly modeled and relevant simulated interactive structures were obtained. The final simulated tetraprotein complex (SoxYZCD) from docked SoxYZ and SoxCD complexes was disclosed to be a highly interactive one with predominant ionic residues. Free energy of folding, solvent accessibility, and conformational shifts (coil-like conformation to helices and sheets) were observed in SoxYZ complex after interacting with SoxCD. The stability of the complex (SoxYZCD) after simulation was also observed through the electrostatic surface potential values. These evaluations were rationalized via biostatistics. This aids SoxCD for recycling SoxY along with thiosulphate, which remains interconnected by four H-bonds with SoxY. Therefore, this novel exploration is endowed with the detailed molecular viewpoint for maintaining the sulphur cycle (globally) including the ocean belts.

Glutamate Gated Proton-Coupled Electron Transfer Activity of a [NiFe]-Hydrogenase.

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. While electron transfer to and from the active site is understood to occur through iron-sulfur clusters, the mechanism of proton transfer is still debated. Two mechanisms for proton exchange with the active site have been proposed that involve distinct and conserved ionizable amino acid residues, one a glutamate, and the other an arginine. To examine the potential role of the conserved glutamate on active site acid-base chemistry, we mutated the putative proton donor E17 to Q in the soluble hydrogenase I from Pyrococcus furiosus using site directed mutagenesis. FTIR spectroscopy, sensitive to the CO and CN ligands of the active site, reveals catalytically active species generated upon reduction with H2, including absorption features consistent with the Nia-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Nia-C, indicates the E17Q mutation does not interfere with the hydride photolysis process generating known intermediates Nia-I1 and Nia-I2. Strikingly, the E17Q mutation disrupts obligatory proton-coupled electron transfer from the Nia-I1 state, thereby preventing formation of Nia-S. These results directly establish E17 as a proton donor/acceptor in the Nia-S ↔ Nia-C equilibrium.

Investigating the Reaction Mechanism of F420-Dependent Glucose-6-phosphate Dehydrogenase from Mycobacterium tuberculosis: Kinetic Analysis of the Wild-Type and Mutant Enzymes.

F420-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, using F420 cofactor as the hydride transfer acceptor, within mycobacteria. A previous crystal structure of wild-type FGD led to a proposed mechanism suggesting that the active site residues His40, Trp44, and Glu109 could be involved in catalysis. We have characterized the wild-type FGD and five FGD variants (H40A, W44F, W44Y, W44A, and E109Q) by fluorescence binding assays and steady-state and pre-steady-state kinetic experiments. Compared to wild-type FGD, all the variants had lower binding affinities for F420, thus suggesting that Trp44, His40, and Glu109 aid in F420 binding. While all the variants had decreased catalytic efficiencies, FGD H40A and W44A were the least efficient, having lost ∼1000- and ∼2000-fold activity, respectively. This confirms a crucial catalytic role for His40 in the FGD reaction and suggests that aromaticity at residue 44 aids catalysis. To investigate the proposed roles of Glu109 and His40 in acid-base catalysis, the pH dependence of kinetic parameters has been determined for the E109Q and H40A mutants and compared to those of the wild-type enzyme. The log kcat-pH profile of wild-type FGD and E109Q revealed two ionizable residues in the enzyme-substrate complex, while H40A displayed only one ionization event. The FGD E109Q variant displayed pH-dependent kinetic cooperativity with respect to the F420 cofactor. The multiple-turnover pre-steady-state kinetics were biphasic for wild-type FGD, W44F, W44Y, and E109Q, while the H40A and W44A variants displayed only a single phase because of their reduced catalytic efficiency.