Electronic aspects of serine protease catalysis

A new electrostatic approach is applied to serine protease catalysis. It is is based upon the demonstration that the polarities, or partial charges, of the atomic components of the molecules involved in the reaction alternate in sign. When the atomic components of opposite polarities of the enzyme and substrate approach close to each other during the catalysis, the electrostatic interactions between them increase in intensity. These increasing interactions are related to the decrease in the energy barrier. When the serine protease--catalyzed reaction is followed from this perspective, it is shown to result in a marked simplification of the catalytic mechanism. A number of concerted proton transfers and electron density displacements around the active site are indicated. This approach is not inconsistent with other electrostatic methods, and is supported by independent partial charge calculations.     

Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase

Using experimentally calibrated density functional calculations on models of the active site of soybean lipoxygenase 1 (SLO-1), insight has been obtained into the coordination flexibility of the iron active site and its molecular mechanism of catalysis. The ferrous form of SLO-1 shows a variation in coordination number in solution that is related to a weakly coordinating Asn694 ligand. From the calculations it is determined that the weak Fe-O(694) bond associated with this coordination flexibility is due to a sideways tilted geometry of Asn694 that is imposed on the site by the protein. Release of this constraint (by altering the hydrogen bonding network) leads to a pure six-coordinate site. In contrast, the ferric form of the enzyme stays five-coordinate. In this case, deprotonation of a coordinated water gives a strong hydroxo donor in the cis position to Asn694, weakening the Fe-O(694) bond. Hence, Asn694 is a stronger ligand to the reduced relative to the oxidized site. Using these experimentally calibrated models, the reaction energy for H-atom transfer in SLO-1 has been calculated to be about -18 kcal/mol. The observed change in coordination number going from five-coordinate in ferric to six-coordinate in ferrous SLO-1 increases the reduction potential of the iron active site. Hence, the protein adjusts the active site for optimal reactivity. Analysis of the electronic structure along the reaction coordinate shows that the H-atom transfer in SLO-1 actually corresponds to a proton-coupled electron transfer (PCET). The transferred electron does not localize on the proton, but tunnels directly from the substrate to the ferric active site in a concerted proton tunneling-electron tunneling (PTET) process. The covalently linked Fe-O-H-C bridge in the transition state lowers the energy barrier and provides an efficient superexchange pathway for this tunneling. The thermal barrier for the PTET process is estimated from the calculations to be about +15 kcal/mol including zero-point energy corrections. This corresponds to a thermal reaction rate of k(therm) approximately 1 s(-1). In comparison, the rate of proton tunneling can be as high as 2 x 10(9) s(-1) under these conditions.     

Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme–substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.     

The mechanism of proton exclusion in the aquaporin-1 water channel

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.     

The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.     

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression—an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression—through fast promoting vibrations, or other means—could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.     

Converting structural changes upon oxidation of cytochrome c to electrostatic reorganization energy

The observed X-ray structural differences between reduced and oxidized cytochrome c are converted to electrostatic energy. This conversion is used to estimate the protein reorganization energy which determines the protein contribution to the activation barrier for the electron transfer reaction. It is shown that the reorganization energy of cytochrome c is much smaller than the corresponding energy for electron transfer in water and that this is consistent with the role for cytochromes as electron transfer catalysts.     

Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase

5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD. Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD. The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1). Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate. The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins. Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state. Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%). The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme. Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence. On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox. In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely. Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox. The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.     

Different interaction modes of two cytochrome-c oxidase soluble CuA fragments with their substrates

Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence CuA center of subunit II. In Thermus thermophilus a soluble cytochrome c552 acts as the electron donor to ba3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c552. In the present study, soluble fragments of the CuA domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c552 and CuA couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Br?nsted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble CuA domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.     

Investigating protein-protein interaction surfaces using a reduced stereochemical and electrostatic model

A method of calculating the electrostatic potential energy between two molecules, using finite difference potential, is presented. A reduced charge set is used so that the interaction energy can be calculated as the two static molecules explore their full six-dimensional configurational space. The energies are contoured over surfaces fixed to each molecule with an interactive computer graphics program. For two crystal structures (trypsin-trypsin inhibitor and anti-lysozyme Fab-lysozyme), it is found that the complex corresponds to highly favourable interacting regions in the contour plots. These matches arise from a small number of protruding basic residues interacting with enhanced negative potential in each case. The redox pair cytochrome c peroxidase-cytochrome c exhibits an extensive favourably interacting surface within which a possible electron transfer complex may be defined by an increased electrostatic complementarity, but a decreased electrostatic energy. A possible substrate transfer configuration for the glycolytic enzyme pair glyceraldehyde phosphate dehydrogenase-phosphoglycerate kinase is presented.     

Hydride transfer catalysed by Escherichia coli and Bacillus subtilis dihydrofolate reductase: coupled motions and distal mutations

This paper reviews the results from hybrid quantum/classical molecular dynamics simulations of the hydride transfer reaction catalysed by wild-type (WT) and mutant Escherichia coli and WT Bacillus subtilis dihydrofolate reductase (DHFR). Nuclear quantum effects such as zero point energy and hydrogen tunnelling are significant in these reactions and substantially decrease the free energy barrier. The donor-acceptor distance decreases to ca 2.7 A at transition-state configurations to enable the hydride transfer. A network of coupled motions representing conformational changes along the collective reaction coordinate facilitates the hydride transfer reaction by decreasing the donor-acceptor distance and providing a favourable geometric and electrostatic environment. Recent single-molecule experiments confirm that at least some of these thermally averaged equilibrium conformational changes occur on the millisecond time-scale of the hydride transfer. Distal mutations can lead to non-local structural changes and significantly impact the probability of sampling configurations conducive to the hydride transfer, thereby altering the free-energy barrier and the rate of hydride transfer. E. coli and B. subtilis DHFR enzymes, which have similar tertiary structures and hydride transfer rates with 44% sequence identity, exhibit both similarities and differences in the equilibrium motions and conformational changes correlated to hydride transfer, suggesting a balance of conservation and flexibility across species.     

Electron density calculations as an extension of protein structure refinement. Streptomyces griseus protease A at 1.5 A resolution

Ab initio quantum mechanical calculations have been used to obtain details of the electron density distribution in a high-resolution refined protein structure. It is shown that with accurate atomic co-ordinates, electron density may be calculated with a quality similar to that which can be obtained directly from crystallographic studies of small organic molecules, and that this density contains information relevant to the understanding of catalysis. Atomic co-ordinates from the 1.8 A and 1.5 A resolution refinements of the crystal structure of protease A from Streptomyces griseus have been used to examine the influence of the environment on the electron density in the side-chain of the active site histidine (His57). The neighbouring aspartic acid 102 is the dominant factor in the environment, and quantum mechanical calculations have been performed on these two residues. Most interesting from the point of view of understanding the catalytic process is the effect that Asp102 has on the electron density in the region of the imidazole nitrogen (N epsilon 2) adjacent to the active site serine 195. In the positively charged imidazolium species, there is a polarization of the N epsilon 2-H bond, reducing the bonding density in a manner that may lower the height of the energy barrier for proton transfer. In the uncharged imidazole species, the proximity of Asp102 causes a movement of density from the lone pair region of the N epsilon 2 into the pi bonding region above and below the plane of the ring. Although it is shown that the primary effect of the aspartic acid is electrostatic, this movement is perpendicular to the direction of the electric field inducing it.     

Toward delineating the structure and function of the particulate methane monooxygenase from methanotrophic bacteria

The particulate methane monooxygenase (pMMO) is a complex membrane protein complex that has been difficult to isolate and purify for biochemical and biophysical characterization because of its instability in detergents used to solubilize the enzyme. In this perspective, we summarize the progress recently made toward obtaining a purified pMMO-detergent complex and characterizing the enzyme in pMMO-enriched membranes. The purified pMMO is a multi-copper protein, with ca. 15 copper ions sequestered into five trinuclear copper clusters: two for dioxygen chemistry and alkane hydroxylation (catalytic or C-clusters) and three to provide a buffer of reducing equivalents to re-reduce the C-clusters following turnover (electron transfer or E-clusters). The enzyme is functional when all the copper ions are reduced. When the protein is purified under ambient aerobic conditions in the absence of a hydrocarbon substrate, only the C-clusters are oxidized; there is an apparent kinetic barrier for electron transfer from the E-cluster copper ions to the C-clusters under these conditions. Evidence is provided in support of both C-clusters participating in the dioxygen chemistry, but only one C-cluster supporting alkane hydroxylation. Acetylene modification of the latter C-cluster in the hydrophobic pocket of the active site lowers or removes the kinetic barrier for electron transfer from the E-clusters to the C-clusters so that all the copper ions could be fully oxidized by dioxygen. A model for the hydroxylation chemistry when a hydrocarbon substrate is bound to the active site of the hydroxylation C-cluster is presented. Unlike soluble methane monooxygenase (sMMO), pMMO exhibits limited substrate specificity, but the hydroxylation chemistry is highly regioselective and stereoselective. In addition, the hydroxylation occurs with total retention of configuration of the carbon center that is oxidized. These results are consistent with a concerted mechanism involving direct side-on insertion of an active singlet "oxene" from the activated copper cluster across the "C-H" bond in the active site. Finally, in our hands, both the purified pMMO-detergent complex and pMMO-enriched membranes exhibit high NADH-sensitive as well as duroquinol-sensitive specific activity. A possible role for the two reductants in the turnover of the enzyme is proposed.     

The catalytic effect of dihydrofolate reductase and its mutants is determined by reorganization energies

The effect of distant mutations on the catalytic reaction of dihydrofolate reductase (DHFR) is reexamined by empirical valence bond simulations. The simulations reproduce for the first time the changes in the observed rate constants (without the use of adjustable parameters for this purpose) and show that the changes in activation barriers are strongly correlated with the corresponding changes in the reorganization energy. The preorganization of the polar groups of enzymes is the key catalytic factor, and anticatalytic mutations destroy this preorganization. Some anticatalytic mutations in DHFR also increase the distance between the donor and acceptor, but this effect is not directly related to catalysis since the native enzyme and the uncatalyzed reaction in water have similar average donor-acceptor distances. Insight into the effect of a mutation is provided by constructing the relevant free energy surfaces in terms of the generalized solute-solvent coordinates. It is shown how the mutations change the reaction coordinate and the activation barrier, and it is clarified that the corresponding changes do not reflect dynamical effects. It is also pointed out that all reactions in a condensed phase involve correlated motions (both in enzymes and in solution) and that the change of such motions upon mutations is a result of the change in the shape of the multidimensional reaction path on the solute-solvent surface, rather than the reason for the change in rate constant. Thus, as far as catalysis is concerned, the change in the activation barrier is due to the change in the electrostatic preorganization energy.     

Pseudogene

Abstract

The article describes organic photochemical reactions in heterogeneous fields. The first part of the article includes an introduction of miscellaneous electrostatic fields adsorbing photoactive species and the second part summarizes the types of photochemical reactions in their fields. Photochemical reactions carried out in various heterogeneous fields, inorganic as well as organic, were classified by their reaction type, that is, unimolecular reactions, bimolecular reactions, energy transfer reactions, and electron transfer reactions.     

Application of electrochemical kinetics to photosynthesis and oxidative phosphorylation: The redox element hypothesis and the principle of parametric energy coupling

It is suggested that the transfer of electrons within the biological electron transfer chain is subject to the laws of electrochemical kinetics, when membrane-bound electron carriers are involved. Consequently, small tightly bound molecular complexes of two or more electron transfer proteins of different redox potential within an energy transducing membrane, which accept electrons from a donor at one membrane surface and donate it to an acceptor at the other, may be regarded as real and functioning molecular redox elements, which convert the free energy of electrons into electrochemical energy. Especially, the transfer of an electron from excited chlorophyll to an electron acceptor can be looked upon as an electrochemical oxidation of excited chlorophyll at such a complex. In this reaction the electron acceptor complex behaves like a polarized electrode, in which the electrochemical potential gradient is provided by a gradient of redox potential of its constituents.Calculations and qualitative considerations show that this concept leads to a consistent understanding of both primary and secondary reactions in photosynthesis (electron capture, delayed light emission, ion transfer, energy conversion) and can also be applied to oxidative phosphorylation. Within the proposed concept, ion transfer and the development of ion gradients have to be considered as results of electrochemical activity—not as intermediates for energy conversion. For energetic reasons, a non steady state, periodic energy coupling mechanism is postulated which functions by periodic changes of the capacity of the (electrochemically) charged energy transducing membrane, during which capacitive surplus energy is released as chemical energy. Energy transducing membranes may thus be considered as electrochemical parametric energy transformers. This concept explains active periodic conformation changes and mechanochemical processes of energy transducing membranes as energetically essential events, which trigger energy conversion according to the principle of variable parameter energy transformers.The electrochemical approach presented here has been suggested and is supported by the observation, that with respect to electron capture and conversion of excitation energy into electrochemical energy, the behaviour of excited chlorophyll at suitable solid state (semiconductor) electrodes is very similar to that of chlorophyll in photosynthetic reaction centers.     

Mutagenesis Alters the Catalytic Mechanism of the Light-driven Enzyme Protochlorophyllide Oxidoreductase

The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.