Helix-capping interaction in lambda Cro protein: a free energy simulation analysis

The stability mutant Tyr-26-->Asp was studied in the Cro protein from bacteriophage lambda using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 +/- 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interaction with the amino terminus of the third alpha-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps--namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macrodipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge-dipole interactions and may be employed to advantage for molecular design.     

The orientation of N-H...O=C and N-H...N hydrogen bonds in biological systems: how good is a point charge as a model for a hydrogen bonding atom?

In order to design new ligands for protein-binding sites of unknown structure, it would be useful to predict the likely sites of hydrogen bonding of an unknown protein fragment to a known molecule. The positions of maxima and minima in the electrostatic potential at appropriate distances from the van der Waals surface were calculated for various small molecules, nucleic acid bases, peptide units and amino acid side chains containing groups which can form the biologically important N-H...O=C and N-H...N hydrogen bonds. Their ability to predict the positions of H and O/N in hydrogen bonded complexes, as predicted by optimising the electrostatic interactions of pairs of such molecules constrained by the molecular shapes, was assessed. It is shown that extrema in the electrostatic potential around the isolated molecules give worthwhile predictions for the locations of hydrogen binding partners. For molecules bound by a single N-H...O=C hydrogen bond, the electrostatic maximum associated with the H is usually less than 1 A from an acceptor atom, while a C=O electrostatic minimum is generally less than 1.5 A from the hydrogen bond proton. However, a significant number of hydrogen bonds form to the opposite lone pair from the electrostatic minimum, in which case the separation is up to 3.3 A. This reflects the broad electrostatic potential well around a carbonyl oxygen between the lone pair directions. The model predicts when neighbouring atoms drastically change the hydrogen bonding characteristics of an N-H or C=O group. Although the geometries of hydrogen bonded complexes are influenced by the other van der Waals contacts between the molecules, particularly multiple hydrogen bonds, these influences are constant when considering hydrogen bonding to a specific uncharacterised binding site. Hence, the consideration of sterically accessible electrostatic extrema will be useful in the design of new ligands.     

Heuristic lipophilicity potential for computer-aided rational drug design: optimizations of screening functions and parameters

In this research we test and compare three possible atom-based screening functions used in the heuristic molecular lipophilicity potential (HMLP). Screening function 1 is a power distance-dependent function, bi/[formula: see text] Ri-r [formula: see text] gamma, screening function 2 is an exponential distance-dependent function, bi exp(-[formula: see text] Ri-r [formula: see text]/d0), and screening function 3 is a weighted distance-dependent function, sign(bi) exp[-xi [formula: see text] Ri-r [formula: see text]/magnitude of bi)]. For every screening function, the parameters (gamma, d0, and xi) are optimized using 41 common organic molecules of 4 types of compounds: aliphatic alcohols, aliphatic carboxylic acids, aliphatic amines, and aliphatic alkanes. The results of calculations show that screening function 3 cannot give chemically reasonable results, however, both the power screening function and the exponential screening function give chemically satisfactory results. There are two notable differences between screening functions 1 and 2. First, the exponential screening function has larger values in the short distance than the power screening function, therefore more influence from the nearest neighbors is involved using screening function 2 than screening function 1. Second, the power screening function has larger values in the long distance than the exponential screening function, therefore screening function 1 is effected by atoms at long distance more than screening function 2. For screening function 1, the suitable range of parameter gamma is 1.0 < gamma < 3.0, gamma = 2.3 is recommended, and gamma = 2.0 is the nearest integral value. For screening function 2, the suitable range of parameter d0 is 1.5 < d0 < 3.0, and d0 = 2.0 is recommended. HMLP developed in this research provides a potential tool for computer-aided three-dimensional drug design.     

Enhancing security and improving interoperability in healthcare information systems

A dataset of 82 protein-ligand complexes of known 3D structure and binding constant Ki was analysed to elucidate the important factors that determine the strength of protein-ligand interactions. The following parameters were investigated: the number and geometry of hydrogen bonds and ionic interactions between the protein and the ligand, the size of the lipophilic contact surface, the flexibility of the ligand, the electrostatic potential in the binding site, water molecules in the binding site, cavities along the protein-ligand interface and specific interactions between aromatic rings. Based on these parameters, a new empirical scoring function is presented that estimates the free energy of binding for a protein-ligand complex of known 3D structure. The function distinguishes between buried and solvent accessible hydrogen bonds. It tolerates deviations in the hydrogen bond geometry of up to 0.25 A in the length and up to 30 degrees in the hydrogen bond angle without penalizing the score. The new energy function reproduces the binding constants (ranging from 3.7 x 10(-2) M to 1 x 10(-14) M, corresponding to binding energies between -8 and -80 kJ/mol) of the dataset with a standard deviation of 7.3 kJ/mol corresponding to 1.3 orders of magnitude in binding affinity. The function can be evaluated very fast and is therefore also suitable for the application in a 3D database search or de novo ligand design program such as LUDI. The physical significance of the individual contributions is discussed.     

Molecular electronic properties of a series of 4-quinolinecarbinolamines define antimalarial activity profile

A detailed computational study on a series of 4-quinolinecarbinolamine antimalarials was performed using the semiempirical Austin model 1 (AM1) quantum chemical method to correlate the electronic features with antimalarial activity and to illuminate more completely the fundamental molecular level forces that affect the function and utility of the compounds. Ab initio (3-21G level) calculations were performed on mefloquine, the lead compound in this series, to check the reliability of the AM1 method. Electron density in specific regions of the molecules appears to play the pivotal role toward activity. A large laterally extended negative potential in the frontal portion of the nitrogen atom of the quinoline ring and the absence of negative potential over the molecular plane are crucial for the potent antimalarials. These electrostatic features are likely to be the modulator of hydrophobicity or lipophilicity of the compounds and, hence, determine their activities. The magnitude of the positive potential located by the hydroxyl hydrogen atom also correlates with potent antimalarial activity. Two negative potential regions occur near the hydroxyl oxygen and piperidyl nitrogen atoms. The two negative potential regions and the positive potential located by the hydroxyl hydrogen atom are consistent with intermolecular hydrogen bonding with the cellular effectors. The present modeling study should aid in efficient designing of this class of antimalarial agents.     

Synthesis, biochemical evaluation, and classical and three-dimensional quantitative structure-activity relationship studies of 7-substituted-1,2,3,4-tetrahydroisoquinolines and their relative affinities toward phenylethanolamine N-methyltransferase and the alpha2-adrenoceptor

7-Substituted-1,2,3,4-tetrahydroisoquinolines (7-substituted-THIQs) are potent inhibitors of phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28), the enzyme involved in the biosynthesis of epinephrine. Unfortunately, most of these compounds also exhibit strong affinity for the alpha2-adrenoceptor. To design a selective (PNMT vs alpha2-adrenoceptor affinity) inhibitor of PNMT, the steric and electrostatic factors responsible for PNMT inhibitory activity and alpha2-adrenoceptor affinity were investigated by evaluating a number of 7-substituted-THIQs. A classical quantitative structure-activity relationship (QSAR) study resulted in a three-parameter equation for PNMT (PNMT pKi = 0.599pi - 0.0725MR + 1. 55sigmam + 5.80; n = 27, r = 0.885, s = 0.573) and a three-parameter equation for the alpha2-adrenoceptor (alpha2 pKi = 0.599pi - 0. 0542MR - 0.951sigmam + 6.45; n = 27, r = 0.917, s = 0.397). These equations indicated that steric effects and lipophilicity play a similar role at either active site but that electronic effects play opposite roles at either active site. Two binding orientations for the THIQs were postulated such that lipophilic and hydrophilic 7-substituents would not occupy the same region of space at either binding site. Using these two binding orientations, based on the lipophilicity of the 7-substituent, comparative molecular field analysis (CoMFA) models were developed that showed that the steric and electrostatic interactions at both sites were similar to those previously elaborated in the QSAR analyses. Both the QSAR and the CoMFA analyses showed that the steric interactions are similar at the PNMT active site and at the alpha2-adrenoceptor and that the electrostatic interactions were different at the two sites. This difference in electrostatic interactions might be responsible for the selectivity of THIQs bearing a nonlipophilic electron-withdrawing group at the 7-position. These QSAR and CoMFA results will be useful in the design of potent and selective (PNMT vs alpha2-adrenoceptor affinity) inhibitors of PNMT.     

A role for CH...O interactions in protein-DNA recognition

The concept of CH...O hydrogen bonds has recently gained much interest, with a number of reports indicating the significance of these non-classical hydrogen bonds in stabilizing nucleic acid and protein structures. Here, we analyze the CH...O interactions in the protein-DNA interface, based on 43 crystal structures of protein-DNA complexes. Surprisingly, we find that the number of close intermolecular CH...O contacts involving the thymine methyl group and position C5 of cytosine is comparable to the number of protein-DNA hydrogen bonds involving nitrogen and oxygen atoms as donors and acceptors. A comprehensive analysis of the geometries of these close contacts shows that they are similar to other CH...O interactions found in proteins and small molecules, as well as to classical NH...O hydrogen bonds. Thus, we suggest that C5 of cytosine and C5-Met of thymine form relatively weak CH...O hydrogen bonds with Asp, Asn, Glu, Gln, Ser, and Thr, contributing to the specificity of recognition. Including these interactions, in addition to the classical protein-DNA hydrogen bonds, enables the extraction of simple structural principles for amino acid-base recognition consistent with electrostatic considerations.     

Molecular lipophilicity potential (MLP) in drug design. Complementing theoretical tools with experimental results

The Molecular Lipophilicity Potential (MLP) is a versatile tool in drug design whose present state and potential developments are reviewed here. The MLP offers a three-dimensional representation of lipophilicity as calculated from partition coefficients. The intermolecular recognition forces and intramolecular effects encoded in lipophilicity are presented, followed by the theoretical foundations and validation of the MLP. It is then demonstrated that the MLP allows for the first time to investigate the dependence of lipophilicity on conformation. As a matter of fact, the MLP combined with an exploration of the conformational space of a solute reveals its "chameleonic" behaviour, i.e. its capacity to adapt to the molecular environment. Other applications of the MLP are presented and illustrated, namely its integration into 3D-QSAR (Comparative Molecular Field Analysis, CoMFA) and its interest as a docking tool.     

Delivery of dehydroepiandrosterone to premenopausal women: effects of micronization and nonoral administration

Previous molecular mechanics calculations suggest that strands of peptide nucleic acids (PNAs) and complementary oligonucleotides form antiparallel duplexes stabilized by interresidue hydrogen bonds. In the computed structures, the amide carbonyl oxygen nearest the nucleobase (O7') forms an interresidue hydrogen bond with the backbone amide proton of the following residue, (n + 1)H1'. Of the 10 published two dimensional 1H NMR structures of a hexameric PNA.RNA heteroduplex. PNA(GAACTC).r(GAGUUC), 9 exhibit two to five potential interresidue hydrogen bonds. In our minimized average structure, created from the coordinates of these 10 NMR structures, three of the five possible interresidue hydrogen bond sites within the PNA backbone display the carbonyl oxygen (O7') and the amide proton (n + 1)H1' distances and N1'-H1'-(n - 1)O7' angles optimal for hydrogen bond formation. The finding of these interresidue hydrogen bonds supports the results of our previous molecular mechanics calculations.     

Binding of the estrogen receptor to DNA. The role of waters

Molecular dynamics simulations are carried out to investigate the binding of the estrogen receptor, a member of the nuclear hormone receptor family, to specific and non-specific DNA. Two systems have been simulated, each based on the crystallographic structure of a complex of a dimer of the estrogen receptor DNA binding domain with DNA. One structure includes the dimer and a consensus segment of DNA, ds(CCAGGTCACAGTGACCTGG); the other structure includes the dimer and a nonconsensus segment of DNA, ds(CCAGAACACAGTGACCTGG). The simulations involve an atomic model of the protein-DNA complex, counterions, and a sphere of explicit water with a radius of 45 A. The molecular dynamics package NAMD was used to obtain 100 ps of dynamics for each system with complete long-range electrostatic interactions. Analysis of the simulations revealed differences in the protein-DNA interactions for consensus and nonconsensus sequences, a bending and unwinding of the DNA, a slight rearrangement of several amino acid side chains, and inclusion of water molecules at the protein-DNA interface region. Our results indicate that binding specificity and stability is conferred by a network of direct and water mediated protein-DNA hydrogen bonds. For the consensus sequence, the network involves three water molecules, residues Glu-25, Lys-28, Lys-32, Arg-33, and bases of the DNA. The binding differs for the nonconsensus DNA sequence in which case the fluctuating network of hydrogen bonds allows water molecules to enter the protein-DNA interface. We conclude that water plays a role in furnishing DNA binding specificity to nuclear hormone receptors.     

Enhancing the relaxivity of paramagnetic coordination complexes through the optimization of the molecular electrostatic potential

The low relaxivity of paramagnetic coordination complexes limits their use as contrast agents in magnetic resonance imaging (MRI). To address this problem, we study the relationship between the molecular structure of these complexes and their relaxivity. While others have investigated the vibrational modes as molecular determinants of the electronic spin relaxation time, we focus on the analysis of the molecular electrostatic potential (MEP) of the paramagnetic coordination complex. Electrostatic forces dominate the interaction between the coordination complex and water. Hence, in addition to steric forces, the molecular electrostatic potential should be a determinant of the lifetime of the water-metal link (tm), the internuclear distance between the water hydrogens and the metal (R), and the number of water molecules attached to the metal in the inner and outer spheres of coordination. We compute the molecular electrostatic potential for a series of model metalloporphyrins because their physical and biologic properties are well known, and they are putative magnetic resonance imaging contrast agents with affinity to neoplastic tissue. Replacing the sulfonato groups in MnTPPS4 with carboxylate groups in the ortho position of the phenyl rings attached to the meso carbons results in an electrostatic focusing field that should reduce R and increase tm. Similar substitutions involving polar groups, including one modeled after a well-known picket-fence porphyrin, are not strong enough to generate a focusing field. Instead, these polar groups should modulate the water-metal interactions through steric interactions. Molecular dynamic simulations show a large outer sphere of coordination around the paramagnet that extends almost three times the distance of the inner sphere of coordination.     

Knowledge-based modeling of a legume lectin and docking of the carbohydrate ligand: the Ulex europaeus lectin I and its interaction with fucose

Ulex europaeus isolectin I is specific for fucose-containing oligosaccharide such as H type 2 trisaccharide alpha-L-Fuc (1-->2) beta-D-Gal (1-->4) beta-D-GlcNAc. Several legume lectins have been crystallized and modeled, but no structural data are available concerning such fucose-binding lectin. The three-dimensional structure of Ulex europaeus isolectin I has been constructed using seven legume lectins for which high-resolution crystal structures were available. Some conserved water molecules, as well as the structural cations, were taken into account for building the model. In the predicted binding site, the most probable locations of the secondary hydroxyl groups were determined using the GRID method. Several possible orientations could be determined for a fucose residue. All of the four possible conformations compatible with energy calculations display several hydrogen bonds with Asp-87 and Ser-132 and a stacking interaction with Tyr-220 and Phe-136. In two orientations, the O-3 and O-4 hydroxyl groups of fucose are the most buried ones, whereas two other, the O-2 and O-3 hydroxyl groups are at the bottom of the site. Possible docking modes are also studied by analysis of the hydrophobic and hydrophilic surfaces for both the ligand and the protein. The SCORE method allows for a quantitative evaluation of the complementarity of these surfaces, on the basis of molecular lipophilicity calculations. The predictions presented here are compared with known biochemical data.     

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.     

Conformational study on trans- and cis-N-acetyl-N'-methylamides of Pro-Xaa dipeptides

Conformational free energy calculations using an empirical potential (ECEPP/2) and the hydration shell model were carried out on the N-acetyl-N'-methylamides of Pro-Xaa dipeptides (Xaa = Ala, Leu, Val, Gly, Cys, Met, Phe, Tyr, Asn, Asp, and Ser) with trans and cis peptide bonds preceding proline residue in the unhydrated and hydrated states. As compared with the results obtained by using the earlier version of ECEPP, the values of beta-bend probabilities are doubled. The average calculated population of cis-dipeptide is about 4%, which is close to the abundance obtained from the analysis of X-ray crystal structures of proteins. The beta-bends are the most dominant structures of cis-dipeptides. Type I, usually having intramolecular hydrogen bonds, contributes greatly to the beta-bend conformations of trans- and cis-dipeptides. However, type I beta-bends of cis-dipeptides do not have any hydrogen bonds. By including the hydration, the beta-bend probabilities for trans- and cis-dipeptides decreased, indicating that the interactions of water molecules with a backbone or side-chain may force the dipeptides to be more distorted or extended. In particular, type II is found to be a dominant beta-bend conformation of trans- and cis-Pro-Gly dipeptides in both the unhydrated and hydrated states. In general, the calculated propensities for Pro-Xaa dipeptides to adopt beta-bend conformations are reasonably consistent with available experimental data. From comparing conformations of Pro and Xaa residues in the dipeptides and single residues, we found that inter-residue interactions and hydration are of importance in determining the conformational properties of the Pro-Xaa dipeptide.     

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Glycoxidation in aortic collagen from STZ-induced diabetic rats and its relevance to vascular damage

Molecular dynamics simulations of alpha-lactalbumin were performed under conditions of neutral pH and low pH in order to study the acid-induced molten globule state. Through the use of experimental techniques such as NMR and CD spectroscopy, molten globules have been characterized as being compact intermediates with secondary structure similar to that of the native protein but with tertiary structure that is disordered. The detailed structure of the molten globule state is unknown, however. Through the use of computer simulations we can study the structural changes which occur upon lowering pH. The simulations presented here differ from previous unfolding simulations in two important ways: the electrostatic interactions are treated more accurately than ever before, and artificially high temperatures are not used to force the protein to unfold. Simulations of 880 psec each were run at pH 7 (control simulation) and pH 2. We concentrate on the interesting changes in the tertiary interactions within the protein with lowering of pH. In particular, there is a loss of native tertiary contacts in the beta domain and interdomain region, and a large decrease in interdomain hydrogen bonds.     

Stabilization of protein crystals by electrostatic interactions as revealed by a numerical approach

We developed a novel algorithm to solve numerically the Poisson-Boltzmann equations under a periodic boundary condition. By employing this algorithm to calculate the electrostatic potentials in two different types of protein crystals, a bovine pancreatic trypsin inhibitor (BPTI) orthorhombic crystal and a pig-insulin cubic crystal, the energy contributions of the electrostatic interactions to the crystals' stability were evaluated. At a high ionic strength, the condensed state of proteins in the crystal was stabilized electrostatically compared with that isolated in dilute solution because the attractive electrostatic interactions between neighboring protein molecules overcame the repulsive forces that originated from the same net charges of the equivalent protein molecules. On the other hand, at a low ionic strength the electrostatic interactions destabilized the crystalline state of both proteins, although a different dependence on the ionic strength was found between them. Here, the insulin crystal was more stable than the BPTI one because of the higher charge density in the BPTI crystal. In all of the solvent ionic strengths investigated, the attractive electrostatic interactions between charge pairs separated by less than 5 A on the respective protein molecules prominently stabilize the protein crystals. Therefore, two protein molecules in the crystals are oriented to compensate each other for their opposite charges on the surfaces. We also found a specific role for bound phosphate ions in the stabilization of the BPTI crystal, based on comparison of the electrostatic energies of the two crystals with and without the ions. By determining the contribution of each atomic charge in the crystals to the electrostatic energy, it was revealed that several electrostatic pairs specifically contributed to the crystal's stability. On the basis of our numerical calculation results, we propose a new method to design protein molecules that adopt stable crystals by replacing destabilizing residues with stabilizing ones and by introducing specific hydrogen bonds or salt bridges between neighboring protein molecules.     

Effects of hydrogen bonds on the redox potential and electronic structure of the bacterial primary electron donor

The primary donor, P, of photosynthetic bacterial reaction centers (RCs) is a dimer of excitonically interacting bacteriochlorophyll (BChl) molecules. The two constituents are named PL and PM to designate their close association with the L- and M-subunits, respectively, of the RC protein. A series of site-directed mutants of RCs from Rhodobacter sphaeroides has been constructed in order to model the effects of hydrogen bonding on the redox midpoint potential and electronic structure of P. The leucine residue at position M160 was genetically replaced with eight other amino acid residues capable of donating a hydrogen bond to the C9 keto carbonyl group of the PM BChl a molecule of P. Fourier transform (FT) (pre)resonance Raman spectroscopy with 1064 nm excitation was used to (i) determine the formation and strengths of hydrogen bonds on this latter keto carbonyl group in the reduced, neutral state (PO), and (ii) determine the degree of localization of the positive charge on one of the two constituent BChl molecules of P in its oxidized, radical cation state (P*+). A correlation was observed between the strength of the hydrogen bond and the increase in PO/P*+ redox midpoint potential. This correlation is less pronounced than that observed for another series of RC mutants where hydrogen bonds to the four pi-conjugated carbonyl groups of P were broken or formed uniquely involving histidinyl residues [Mattioli, T. A., Lin, X., Allen, J. P. and Williams, J. C. (1995) Biochemistry 34, 6142-6152], indicating that histidinyl residues are more effective in raising the PO/P*+ redox midpoint potential via hydrogen bond formation than are other hydrogen bond-forming residues. In addition, an increase in positive charge localization is correlated with the strength of the hydrogen bond and with the PO/P*+ redox midpoint potential. This latter correlation was analyzed using an asymmetric bacteriochlorophyll dimer model based on Hückel-type molecular orbitals in order to obtain estimates of certain energetic parameters of the primary donor. Based on this model, the correlation is extrapolated to the case of complete localization of the positive charge on PL and gives a predicted value for the P/P+ redox midpoint potential similar to that experimentally determined for the Rb. sphaeroides HL(M202) heterodimer. The model yields parameters for the highest occupied molecular orbital energies of the two BChl a constituents of P which are typical for the oxidation potential of isolated BChl a in vitro, suggesting that the protein, as compared to many solvents, does not impart atypical redox properties to the BChl a constituents of P.