Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or “site.” Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment–proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment–protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna–Mathews–Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.     

How proteins trigger excitation energy transfer in the FMO complex of green sulfur bacteria

A simple electrostatic method for the calculation of optical transition energies of pigments in protein environments is presented and applied to the Fenna-Matthews-Olson (FMO) complex of Prosthecochloris aestuarii and Chlorobium tepidum. The method, for the first time, allows us to reach agreement between experimental optical spectra and calculations based on transition energies of pigments that are calculated in large part independently, rather than fitted to the spectra. In this way it becomes possible to understand the molecular mechanism allowing the protein to trigger excitation energy transfer reactions. The relative shift in excitation energies of the seven bacteriochlorophyll-a pigments of the FMO complex of P. aestuarii and C. tepidum are obtained from calculations of electrochromic shifts due to charged amino acids, assuming a standard protonation pattern of the protein, and by taking into account the three different ligand types of the pigments. The calculations provide an explanation of some of the earlier results for the transition energies obtained from fits of optical spectra. In addition, those earlier fits are verified here by using a more advanced theory of optical spectra, a genetic algorithm, and excitonic couplings obtained from electrostatic calculations that take into account the influence of the dielectric protein environment. The two independent calculations of site energies strongly favor one of the two possible orientations of the FMO trimer relative to the photosynthetic membrane, which were identified by electron microscopic studies and linear dichroism experiments. Efficient transfer of excitation energy to the reaction center requires bacteriochlorophylls 3 and 4 to be the linker pigments. The temporal and spatial transfer of excitation energy through the FMO complex is calculated to proceed along two branches, with transfer times that differ by an order of magnitude.     

Identification of key stabilizing interactions of amyloid-β oligomers based on fragment molecular orbital calculations on macrocyclic β-hairpin peptides

Analyzing the electronic states and inter-/intra-molecular interactions of amyloid oligomers expand our understanding of the molecular basis of Alzheimer's disease and other amyloid diseases. In the current study, several high-resolution crystal structures of oligomeric assemblies of Aβ-derived peptides have been studied by the ab initio fragment molecular orbital (FMO) method. The FMO method provides comprehensive details of the molecular interactions between the residues of the amyloid oligomers at the quantum mechanical level. Based on the calculations, two sequential aromatic residues (F19 and F20) and negatively charged E22 on the central region of Aβ have been identified as key residues in oligomer stabilization and potential interesting pharmacophores for preventing oligomer formation.     

Molecular Cloning,Sequence, and Expression of Mouse Flavin-Containing Monooxygenases 1 and 5 (FMO1 and FMO5)

Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5′-flanking region, and 413 in the 3′-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5′-flanking region, and 757 in the 3′-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83–94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83–84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward n -octylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 205–212, 1998     

Effect of Zn ion on the structure and electronic states of Aβ nonamer: molecular dynamics and ab initio molecular orbital calculations

Aggregates of amyloid-beta proteins (Aβ) have been recognised to be intimately related to pathogenesis of Alzheimer’s disease (AD). Indeed, Aβ aggregates of various sizes from dimers to fibrils were found in the brains of AD patients, and these aggregates can be self-organised. Since abnormal accumulation of metal ions such as Zn, Cu and Fe was also observed in the brains, the association between Aβ aggregations and these metal ions has been studied widely. In the present study, to elucidate the influence of Zn ions on the stability of Aβ aggregates, we performed molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the Aβ nonamers with and without Zn ions and investigated the change in its structure and electronic states induced by Zn ions at atomic and electronic levels. The MD simulations revealed that Aβ nonamer cannot keep its symmetry structure, whereas Aβ nonamer with Zn ions keeps the structure. The FMO results indicated that electrostatic interactions among the charged amino-acid residues of Aβ nonamer are significantly changed by the influence of Zn ions to stabilise Aβ nonamer. These results provide useful information for proposing novel compounds, which binds specifically to Aβ and inhibits the Aβ aggregation.     

Hydrogen-deuterium exchange mass spectrometry reveals the interaction of Fenna-Matthews-Olson protein and chlorosome CsmA protein

In green-sulfur bacterial photosynthesis, excitation energy absorbed by a peripheral antenna structure known as the chlorosome is sequentially transferred through a baseplate protein to the Fenna-Matthews-Olson (FMO) antenna protein and into the reaction center, which is embedded in the cytoplasmic membrane. The molecular details of the optimized photosystem architecture required for efficient energy transfer are only partially understood. We address here the question of how the baseplate interacts with the FMO protein by applying hydrogen/deuterium exchange coupled with enzymatic digestion and mass spectrometry analysis to reveal the binding interface of the FMO antenna protein and the CsmA baseplate protein. Several regions on the FMO protein, represented by peptides consisting of 123-129, 140-149, 150-162, 191-208, and 224-232, show significant decreases of deuterium uptake after CsmA binding. The results indicate that the CsmA protein interacts with the Bchl a #1 side of the FMO protein. A global picture including peptide-level details for the architecture of the photosystem from green-sulfur bacteria can now be drawn.     

Reassessment of Hammett σ as an effective parameter representing intermolecular interaction energy-links between traditional and modern QSAR approaches

The Hammett σ constant has for a long time been known to be one of most important linear free-energy related parameters that correlate with biological activity. It is a conventionally used electronic parameter in studies of enzymatic quantitative structure-activity relationships (QSAR). However, it is not necessarily obvious why σ represents variations in the free-energy change associated with the complex formation between a congeneric series of ligands with their target protein. So far, several powerful molecular calculations, such as the ab initio fragment molecular orbital (FMO) one, that are directly applicable to ligand-protein complexes have emerged. In this study, we comprehensively reevaluate experimentally derived parameter σ confirming it represents intermolecular interaction energy terms, by applying molecular orbital (MO) calculations to a simple ligand-protein complex model. The current results provide a rational and quantitative basis for bridging the gap between the traditional QSAR approach and 'the modern QSAR one', which involves the molecular calculations to evaluate the overall free-energy change for complex formation.     

Potential roles of flavin-containing monooxygenases in sulfoxidation reactions of l-methionine, N-acetyl-l-methionine and peptides containing l-methionine

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxidation of a large number of sulfur-, selenium-, and nitrogen-containing compounds. Five active isoforms (FMO1-5) have been identified and shown to be differently expressed in various mammalian tissues. Previous work from this laboratory has shown l-methionine to be S-oxidized by rat, rabbit and human FMO1-4, with FMO3 exhibiting the highest stereoselectivity for the formation of the d-diastereomer of methionine sulfoxide. In this report, we describe new studies aimed at determining if N-acetyl-l-methionine and peptides containing l-methionine can be substrates for FMOs. Experiments were carried out using either rabbit liver microsomes or human cDNA-expressed FMOs. The results show that while N-acetyl-l-methionine and peptides with a modified methionine amino group may not function as substrates for FMOs, peptides containing a free N-terminal methionine may act as FMO substrates. With human cDNA-expressed FMO1, FMO3, and FMO5, both FMO1 and FMO3 exhibited activity with the active peptides whereas FMO5 was inactive. With FMO3, the activity measured with methionine was similar (1 mM) or higher (5 mM) than the activity measured with H-Met-Val-OH and H-Met-Phe-OH. With FMO1, H-Met-Phe-OH and methionine exhibited similar activities whereas activity with H-Met-Val-OH was much lower. Collectively, the results show that FMOs can oxidize peptides containing a free N-terminal methionine. Thus, the role of FMOs in the oxidation of methionine in larger peptides or proteins warrants further investigation.     

Native electrospray mass spectrometry reveals the nature and stoichiometry of pigments in the FMO photosynthetic antenna protein

The nature and stoichiometry of pigments in the Fenna-Matthews-Olson (FMO) photosynthetic antenna protein complex were determined by native electrospray mass spectrometry. The FMO antenna complex was the first chlorophyll-containing protein that was crystallized. Previous results indicate that the FMO protein forms a trimer with seven bacteriochlorophyll a in each monomer. This model has long been a working basis to understand the molecular mechanism of energy transfer through pigment/pigment and pigment/protein coupling. Recent results have suggested, however, that an eighth bacteriochlorophyll is present in some subunits. In this report, a direct mass spectrometry measurement of the molecular weight of the intact FMO protein complex clearly indicates the existence of an eighth pigment, which is assigned as a bacteriochlorophyll a by mass analysis of the complex and HPLC analysis of the pigment. The eighth pigment is found to be easily lost during purification, which results in its partial occupancy in the mass spectra of the intact complex prepared by different procedures. The results are consistent with the recent X-ray structural models. The existence of the eighth bacteriochlorophyll a in this model antenna protein gives new insights into the functional role of the FMO protein and motivates the need for new theoretical and spectroscopic assignments of spectral features of the FMO protein.     

Identification of a flavin-monooxygenase as the S-oxygenating enzyme in aliphatic glucosinolate biosynthesis in Arabidopsis

The cancer-preventive activity of cruciferous vegetables is commonly attributed to isothiocyanates resulting from the breakdown of the natural products glucosinolates (GSLs). Sulforaphane, the isothiocyanate derived from 4-methylsulfinylbutyl GSL, is thought to be the major agent conferring cancer-preventive properties, whereas the isothiocyanate of 4-methylthiobutyl GSL does not have the same activity. We report the identification of an Arabidopsis flavin-monooxygenase (FMO) enzyme, FMO(GS-OX1), which catalyzes the conversion of methylthioalkyl GSLs into methylsulfinylalkyl GSLs. This is evidenced by biochemical characterization of the recombinant protein, and analyses of the GSL content in FMO(GS-OX1) overexpression lines and an FMO(GS-OX1) knock-out mutant of Arabidopsis. The FMO(GS-OX1) overexpression lines show almost complete conversion of methylthioalkyl into methylsulfinylalkyl GSLs, with an approximately fivefold increase in 4-methylsulfinylbutyl GSL in seeds. Identification of FMO(GS-OX1) provides a molecular tool for breeding of Brassica vegetable crops with increased levels of this important GSL, which has implications for production of functional foods enriched with the cancer-preventive sulforaphane.     

A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.     

Frontier molecular orbital analysis of Cu(n)-O(2) reactivity

Frontier molecular orbital (FMO) theory coupled with density functional calculations has been applied to investigate the chemical reactivity of three key bioinorganic Cu(n)-O(2) complexes, the mononuclear end-on hydroperoxo-Cu(II), the side-on bridged mu-eta(2):eta(2)-O(2)(2-) Cu(II)(2) dimer and the bis-mu-oxo Cu(III)(2) dimer. Two acceptor orbitals (sigma* and pi*) of each complex and two types of donating substrates (sigma-substrate, phosphine; pi-substrate, alkylbenzene) are considered in the electrophilic attack mechanism. The angular dependences of different reaction pathways are determined using FMO theory and the angular overlap model. Including steric effects, the sigma*/sigma and pi*/pi pathways are found more reactive than the corresponding cross sigma*/pi and pi*/sigma pathways which have poor donor-acceptor orbital overlaps in the sterically constrained substrate access region.     

Functional characterization of genetic variants of human FMO3 associated with trimethylaminuria

Impaired conversion of trimethylamine to trimethylamine N-oxide by human flavin containing monooxygenase 3 (FMO3) is strongly associated with primary trimethylaminuria, also known as 'fish-odor' syndrome. Numerous non-synonymous mutations in FMO3 have been identified in patients suffering from this metabolic disorder (e.g., N61S, M66I, P153L, and R492W), but the molecular mechanism(s) underlying the functional deficit attributed to these alleles has not been elucidated. The purpose of the present study was to determine the impact of these disease-associated genetic variants on FMO3 holoenzyme formation and on steady-state kinetic parameters for metabolism of several substrates, including trimethylamine. For comparative purposes, several common allelic variants not associated with primary trimethylaminuria (i.e., E158K, V257M, E308G, and the E158K/E308G haplotype) were also analyzed. When recombinantly expressed in insect cells, only the M66I and R492W mutants failed to incorporate/retain the FAD cofactor. Of the remaining mutant proteins P153L and N61S displayed substantially reduced (<10%) catalytic efficiencies for trimethylamine N-oxygenation relative to the wild-type enzyme. For N61S, reduced catalytic efficiency was solely a consequence of an increased K(m), whereas for P153L, both K(m) and k(cat) were altered. Similar results were obtained when benzydamine N-oxygenation was monitored. A homology model for FMO3 was constructed based on the crystal structure for yeast FMO which places the N61 residue alone, of the mutants analyzed here, in close proximity to the FAD catalytic center. These data demonstrate that primary trimethylaminuria is multifactorial in origin in that enzyme dysfunction can result from kinetic incompetencies as well as impaired assembly of holoprotein.     

Pump-probe anisotropies of Fenna-Matthews-Olson protein trimers from Chlorobium tepidum: a diagnostic for exciton localization?

Exciton calculations on symmetric and asymmetric Fenna-Matthews-Olson (FMO) trimers, combined with absorption difference anisotropy measurements on FMO trimers from the green bacterium Chlorobium tepidum, suggest that real samples exhibit sufficient diagonal energy disorder so that their laser-excited exciton states are noticeably localized. Our observed anisotropies are clearly inconsistent with 21-pigment exciton simulations based on a threefold-symmetric FMO protein. They are more consistent with a 7-pigment model that assumes that the laser-prepared states are localized within a subunit of the trimer. Differential diagonal energy shifts of 50 cm(-1) between symmetry-related pigments in different subunits are large enough to cause sharp localization in the stationary states; these shifts are commensurate with the approximately 95 cm(-1) inhomogeneous linewidth of the lowest exciton levels. Experimental anisotropies (and by implication steady-state linear and circular dichroism) likely arise from statistical averaging over states with widely contrasting values of these observables, in consequence of their sensitivity to diagonal energy disorder.     

Molecular recognition mechanism of FK506 binding protein: an all-electron fragment molecular orbital study

The fragment molecular orbital (FMO) method has enabled electronic structure calculations and geometry optimizations of very large molecules with ab initio quality. We applied the method to four FK506 binding protein (FKBP) complexes (denoted by their PDB codes 1fkb, 1fkf, 1fkg, and 1fki) containing rapamycin, FK506, and two synthetic ligands. The geometries of reduced complex models were optimized at the restricted Hartree–Fock (FMO‐RHF) level using the 3‐21G basis set, and then for a better estimate of binding, the energetics were refined at a higher level of theory (2nd order Møller–Plesset perturbation theory FMO‐MP2 with the 6‐31G* basis set). Thus, obtained binding energies were ?103.9 (?82.0), ?102.2 (?69.2), ?70.1 (?57.7), and ?71.3 (?55.3) kcal/mol for 1fkb, 1fkf, 1fkg, and 1fki, respectively, where the correlation contribution is given in parentheses. The results show that the electron correlation contribution to binding is extremely important, and it accounts for 70–80% of the binding energy. The molecular recognition mechanism of FKBP was analyzed in detail based on the FMO‐pair interactions between protein residues and the ligands. Solvation effects on the protein–ligand binding were estimated using the Poisson–Boltzmann/surface area model. Proteins 2007. © 2007 Wiley‐Liss, Inc.     

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.     

The flavin-containing monooxygenase in mouse lung: Evidence for expression of multiple forms

The flavin-containing monooxygenase (FMO) was purified from mouse lung microsomes. On SDS-PAGE, the purified enzyme separated as two bands, a major band of 58,000 daltons and a minor band of 59,000 daltons. Antibodies to mouse liver FMO cross-reacted with both bands in the purified preparations, whereas antibodies to rabbit lung FMO cross-reacted only with the major band. In microsomal preparations the major band was recognized by both antibodies, but neither antibody detected the minor band in microsomes. A cDNA encoding the pig liver FMO hybridized with mRNA isolated from mouse liver, kidney, and lung, whereas cDNA encoding the rabbit lung FMO hybridized only with mouse lung and kidney mRNA. Thermal stability studies showed that the FMO preparation purified from mouse lung consisted of a heat-stable and a heat-labile component. The heat-labile component of lung FMO was inhibited competitively by imipramine, whereas the heat-stable component was insensitive to the presence of imipramine. Immunoprecipitation of purified mouse lung FMO with anti-rabbit lung FMO completely removed the protein band reactive to anti-rabbit lung FMO while leaving reactivity to anti-liver FMO. The catalytic and immunochemical differences seen between FMO from rabbit lung and mouse lung appear to result from the expression of at least two forms of FMO in the mouse lung, one similar to the rabbit pulmonary form and one similar to the major mouse liver form of FMO.     

Distinct pulmonary and hepatic forms of flavin-containing monooxygenase in sheep

1. Flavin-containing monooxygenase (FMO) in pulmonary and hepatic microsomes from sheep was analyzed by western blotting by probing with antibodies raised against FMO purified from rabbit lung and pig liver. 2. Pulmonary microsomes from sheep contain a single major protein which cross-reacts with the antibody to rabbit lung FMO, but no band can be observed when probed with the antibody to the pig liver enzyme. Likewise, sheep liver microsomes contain a protein which cross-reacts with the antibody to pig liver FMO, but no significant staining is observed following incubation with antibody to the lung enzyme. 3. Sheep pulmonary and hepatic microsomal FMO also display a difference in activity toward chlorpromazine and n-dodecylamine. 4. Preliminary evidence suggests that sheep FMO may be induced (liver) or repressed (lung) during pregnancy. 5. Sheep are similar to rodents (rat, mouse, guinea pig, hamster and rabbit) in having distinct forms of pulmonary and hepatic FMO. The immunochemical and catalytic difference between sheep liver and lung FMO is similar to that of rabbit.     

Structure-based rational design of novel hit compounds for pyruvate dehydrogenase multienzyme complex E1 components from Escherichia coli

Pyruvate dehydrogenase multienzyme complex (PDHc) E1 component plays a pivotal role in cellular metabolism to convert the product of glycolysis (pyruvate) to acetyl-CoA, and has been reported as a potential target for anti-microbial and herbicide. In present study, based on the thiamin diphosphate (ThDP) site, four novel hit compounds with high inhibitory activity against the PDHc-E1 from Escherichia coli were firstly designed by using structure-based molecular docking methods. As expected, among four compounds, the compound 3a is the best inhibitor by far, with IC₅₀ value of 6.88 μM against PDHc-E1 from E. coli. To elucidate the interaction mechanism between the active site of PDHc-E1 and its inhibitor, the docking-based molecular dynamics simulation (MD) and MD-based ab initio fragment molecular orbital (FMO) calculations were also further performed. The positive results indicated that all modeling strategies presented in the current study most like to be an encouraging way in design of novel lead compounds with structural diversity for PDHc-E1 in the future.