Hydrogen bonding of flavoprotein: II. Effect of hydrogen bonding at hetero atoms of reduced flavin on its reactivity

The effect of hydrogen bonding at hetero atoms of reduced flavin on its reactivity was studied by ab initio molecular orbital calculations. Among the atoms in the isoalloxazine nucleus of lumiflavin, C(4a) was found to be the most reactive with neutral electrophiles such as molecular oxygen, whereas no reactivity of N(5) can be expected, because of its negative charge. The reactivity of C(4a) is markedly enhanced by hydrogen bonding at N(1) and N(3) in a hydrophobic environment, while it is decreased when hydrogen bonding occurs at all the hetero atoms, as in the case of an aqueous solution of flavin.     

Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase

The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.     

Studies in a model system on the effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability

The effect of hydrogen bonding at hetero atoms of oxidized flavin on its electron acceptability was studied by the ab initio molecular orbital method. The calculations were carried out for all possible lumiflavin-H2O complexes and for some lumiflavin-formamide complexes. Calculated data showed that the magnitudes of hydrogen bonding energy at the hetero atoms are in the order of N(3)H greater than N(5) greater than O(12) greater than N(1) greater than O(14). It was found that the atomic orbital coefficient of the lowest unoccupied molecular orbital is the largest at N(5) and that hydrogen bonding at N(1), N(5), O(12), and O(14) increases the electron acceptability of the oxidized flavin at N(5), while hydrogen bonding at N(3)H decreases it.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of transition metal complexes to guanine and guanine–cytosine: hydrogen bonding and covalent effects

Density functional calculations and Atoms in Molecules analysis are used to investigate the role of covalent and hydrogen bondings in determining the binding of transition metal complexes to guanine, and the subsequent effect on pairing with cytosine. Hydrogen bonding is ubiquitous, and typically contributes ca. 10% to overall binding, a value that varies with the coordination site on guanine, as well as metal and ligands. Early transition metals show a clear preference for the O6 position, while later ones prefer N7, the crossover point coming at the vanadium group. Metallation at N7 causes a redistribution of hydrogen bonding strength between guanine and cytosine, but does not greatly affect the overall pairing energy. In contrast, metallation at O6 strongly reduces the pairing energy, as may be expected given the role of O6 in pairing guanine with cytosine. This effect can be quantified using electron density properties, and seems to be due to both electrostatic repulsion from the positive metal centre and a redistribution of electron density within guanine itself. Qualitative agreement with experimental mass spectroscopic results is obtained.     

Enzyme-catalyzed redox reactions with the flavin analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphte, and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'-adenosine ester.

The ability of 5-deazaisoalloxazines to substitute for the isoalloxazine (flavin) coenzyme has been examined with several flavoenzymes. Without exception, the deazaflavin is recognized at the active site and undergoes a redox change in the presence of the specific enzyme substrate. Thus, deazariboflavin is reduced catalytically by NADH in the presence of the Beneckea harveyi NAD(P)H:(flavin) oxidoreductase, the reaction proceeding to an equilibrium with an equilibrium constant near unity. This implies an E0 of -0.310 V for the deazariboflavindihydrodeazariboflavin couple, much lower than that for isoalloxazines. With this enzyme, both riboflavin and deazariboflavin show the same stereospecificity with respect to the pyridine nucleotide, and despite a large difference in Vmax for the two, both have the same rate-determining step (hydrogen transfer). Direct transfer of the hydrogen is seen between the nicotinamide and deazariboflavin in both reaction directions. DeazaFMN reconstituted yeast NADPH: (acceptor) oxidoreductase (Old Yellow Enzyme), and deazaFAD reconstituted D-amino acid:O2 oxidoreductase and Aspergillus niger D-glucose O2 oxidoreductase are all reduced by substrate at approximately 10(-5) the rate of holoenzyme; none are reoxidized by oxygen or any of the tested artificial electron acceptors, though deazaFADH-bound to D-amino acid:O2 oxidoreductase is rapidly oxidized by the imino acid product. Direct hydrogen transfer from substrate to deazaflavin has been demonstrated for both deazaFAD-reconstituted oxidases. These data implicate deazaflavins as a unique probe of flavin catalysis, in that any mechanism for the flavin catalysis must account for the deazaflavin reactivity as well.     

Role of hydrogen bonding in red cell aggregation.

The role of hydrogen bonding in red cell aggregation induced by dextran was studied with the use of urea, an inhibitor for hydrogen bonding. In order to avoid hemolysis of red cells by the high concentration of urea, the studies were performed on human red cells hardened in glutaraldehyde. The degree of red cell aggregation at Hct = 45% was estimated by the use of a coaxial cylinder viscometer. The viscometric aggregation index (VAI) was calculated from viscosity values at shear rates of 52 sec-1 (eta H) and 0.05 sec-1 (eta L); VAI = (eta L - eta H)/eta H. Red cells with surface charge intact and with charge removal by neuraminidase treatment were studied. Urea at high concentrations, e.g., 6 M, significantly inhibited red cell aggregation induced by dextran. These findings indicate that hydrogen bonding plays an important role in dextran-induced red cell aggregation. An understanding of the nature of the forces involved in red cell aggregation serves to establish the physicochemical principles of cell-to-cell interactions induced by macromolecules.     

Resonance Raman studies of ETE dehydrogenase (an iron sulfur flavoprotein)

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100cm-1-1600 cm-1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm-1. The frequency of the band corresponds to the 335 cm-1 band associated with the strongest Fe-S stretching mode in the 4Fe-4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe-3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do.     

Resonance Raman spectra of flavin semiquinones stabilized by N5 methylation

Resonance Raman spectra are reported for the semiquinone of N5-methyl derivatives of FMN (flavin mononucleotide) in H2O and 2H2O, 8-chloro FMN and FAD (flavin adenine dinucleotide) with 647.1 nm excitation, in the first pi-pi absorption band, using KI to quench fluorescence. The spectral pattern is similar to that of oxidized flavin, in its first absorption band, but with appreciable shifts, up to approx. 50 cm-1, in corresponding frequencies. There are also significant shifts with respect to the previously reported resonance Raman spectrum of flavodoxin semiquinone, reflecting the substitution of CH3 for H at N5. The N5-methyl FAD semiquinone spectrum is also reported for 514.5 nm excitation, in resonance with the second pi-pi transition. The intensity pattern is quite different, the spectrum being dominated by a band at 1611 cm-1, assigned to a mode localized primarily on the central pyrazine ring.     

Structural intermediate in the photocycle of a BLUF (sensor of blue light using FAD) protein Slr1694 in a Cyanobacterium Synechocystis sp. PCC6803

Slr1694 in Synechocystis sp. PCC6803 is a family of blue-light photoreceptors based on flavin adenine dinucleotide (FAD) called BLUF (sensor of blue light using FAD) proteins, which include AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis. Illumination of dark-state Slr1694 at 15 degrees C reversibly induced a signaling light state characterized by the red shift in the UV-visible spectrum and by the light-induced Fourier transform infrared (FTIR) difference spectrum for structural changes of a bound flavin and apo protein. Illumination at the medium-low temperature (-35 degrees C) led to the red shift in the UV-visible spectrum despite some small difference in the light-induced changes. In contrast, the -35 degrees C illumination resulted in a completely different light-induced FTIR spectrum, in which almost all of the bands were suppressed with the exception of the bands for the change of C4=O bonding of the FAD isoalloxazine ring. The C4=O bands were induced at -35 degrees C with almost the same intensity, but the band frequency for the light state was upshifted by 6 cm(-)(1). The changes in frequency of the light-state C4=O band and in amplitude of other bands showed the same temperature dependence with a half-change temperature at approximately -20 degrees C. It was indicated that the light-induced structural changes of apo protein and FAD were inhibited at low temperature with the exception of the change in hydrogen bonding to the C4=O group. The light-induced formation of the FTIR bands was similarly inhibited by sample dehydration. We discussed the possibility that this constrained light state is a trapped intermediate state in the photocycle of Slr1694.     

Resonance Raman study of flavins and the flavoprotein fatty acyl coenzyme A dehydrogenase.

The resonance Raman (RR) spectra of FMN, FAD, FAD in D2O, and 7,8-dimethyl-1, 10-ethyleneisoalloxazinium perchlorate have been obtained by employing KI as a collisional fluorescence-quenching agent. The spectra are very similar to those obtained recently by using the CARS technique to eliminate fluorescence. Spectra have also been obtained for several species in which flavin is known to fluoresce only weakly. We report RR spectra of protonated FMN, FMN semiquinone cation, the general fatty acyl-CoA dehydrogenase, and two "charge-transfer" complexes of fatty acyl-CoA dehydrogenase. Tentative assignment of several vibrational bands can be made on the basis of our flavin spectra. RR spectra of fatty acyl-CoA and its complexes are consistent with the previous hypothesis that visible spectral shifts observed during formation of acetoacetyl-CoA and crotonyl-CoA complexes of fatty acyl-CoA dehydrogenase result from charge-transfer interactions in which the ground state is essentially nonbonding as opposed to interactions in which complete electron transfer occurs to form FAD semiquinone. The only significant change in the RR spectrum of FAD on binding to enzyme occurs in the 1250-cm-1 region of the spectrum, a region associated with delta N--H of N-3. The position of this band in fatty acyl-CoA dehydrogenase and the other flavoproteins studied to date is discussed in terms of hydrogen bonding between flavin and protein.     

Crystal structure of oxidized flavodoxin from a red alga Chondrus crispus refined at 1.8 A resolution. Description of the flavin mononucleotide binding site.

In order to describe the detailed conformation of the oxidized flavodoxin from a eukaryotic red alga, Chondrus crispus, the crystal structure has been refined by a restrained least-squares method. The crystallographic R factor is 0.168 for 13,899 reflections with F greater than 2 sigma F between 6.0 and 1.8 A resolution. The refined model includes 173 amino acid residues, flavin mononucleotide (FMN) and 110 water molecules. The root-mean-square deviation in bond lengths from ideal values is 0.015 A, and the mean co-ordinate error is estimated to be 0.2 A. The FMN is located at the periphery of the molecule. The orientation of the isoalloxazine ring is such that the C-7 and C-8 methyl groups are exposed to solvent and the pyrimidine moiety is buried in the protein. Three peptide segments, T8-T13, T55-T58 and D94-C103, are involved in FMN binding. The first segment of T8-T13 enfolds the phosphate group of the FMN. The three oxygen atoms in the phosphate group form extensive hydrogen bonds with amide groups of the main chain and the O gamma atoms of the side-chains in this segment. T55 O and W56 N epsilon 1 in the second segment form hydrogen bonds with O-2 in the ribityl moiety and one of the oxygen atoms in the phosphate group, respectively. The O gamma H of T58 forms a hydrogen bond with the N-5 atom in the isoalloxazine ring, which is expected to be protonated in the semiquinone form. The third segment is in contact with the isoalloxazine ring. It appears that the hydrogen bond acceptor of the NH of Asp94 in the third segment is O-2 rather than N-1 in the isoalloxazine ring. The isoalloxazine ring is flanked by the side-chains of Trp56 and Tyr98; it forms an angle of 38 degrees with the indole ring of Trp56 and is almost parallel to the benzene ring of Tyr98. The environment of the phosphate group is conserved as in other flavodoxins whereas that of the isoalloxazine ring differs. The relationship between the hydrogen bond to the N-5 in the ring and the redox potential for the oxidized/semiquinone couple is discussed.     

Tyrosine B10 triggers a heme propionate hydrogen bonding network loop with glutamine E7 moiety

Propionates, as peripheral groups of the heme active center in hemeproteins have been described to contribute in the modulation of heme reactivity and ligand selection. These electronic characteristics prompted the question of whether the presence of hydrogen bonding networks between propionates and distal amino acids present in the heme ligand moiety can modulate physiological relevant events, like ligand binding association and dissociation activities. Here, the role of these networks was evaluated by NMR spectroscopy using the hemoglobin I PheB10Tyr mutant from Lucina pectinata as model for TyrB10 and GlnE7 hemeproteins. (1)H-NMR results for the rHbICN PheB10Tyr derivative showed chemical shifts of TyrB10 OHη at 31.00ppm, GlnE7N(ε1)H/N(ε2)H at 10.66ppm/-3.27ppm, and PheE11 C(δ)H at 11.75ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. Strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-propionate group, GlnE7/TyrB10 and TyrB10/CN suggest that this hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the heme-ligand moiety.     

Resonance coherent anti-Stokes Raman scattering (CARS) spectra of flavin adenine dinucleotide,riboflavin binding protein and glucose oxidase

Coherent anti-Stokes Raman scattering spectra, in resonance with the isoalloxazine visible electronic transition, have been obtained down to 300 cm^?1 for flavin adenine dinucleotide, riboflavin binding protein and glucose oxidase, in H₂O and D₂O. Several isoalloxazine vibrational modes can be identified by analogy with those of uracil. Of particular interest is a band at ～1255 cm^?1 in H₂O, which is replaced by another at ～1295 cm^?1, in D₂O. The H₂O band appears to be a sensitive monitor of H-bonding of the N3 isoalloxazine proton to a protein acceptor group. It shifts down by 10 cm^?1 in riboflavin binding protein, and disappears altogether in glucose oxidase. Other band shifts, of 3–5 cm^?1, are similar for the two flavoproteins, and may reflect environmental changes between aqueous solution and the protein binding pockets.     

Resonance Raman spectra of carbon-13- and nitrogen-15-labeled riboflavin bound to egg-white flavoprotein.

The resonance Raman spectra of [2-13C]-, [4a-13C]-, [4-13C]-8 [10a-13C]-, [2,4,4a, 10a-13C]-, [5-15N]-, [1,3-15N]-, and [1,3,5-15N]riboflavin bound to egg-white proteins were observed for N(3)-H and N(3)-D forms with spontaneous Raman technique by using the 488.0-nm excitation line of an argon ion laser. The fluorescence of riboflavin was quenched by forming a complex with egg-white riboflavin binding protein. The in-plane displacements of the C(2), C(4a), N(1), N(3), and N(5) atoms during each Raman active vibration were calculated from the observed isotopic frequency shifts. The 1252-cm-1 mode of the N(3)-H form was found to involve large vibrational displacements of the C(2) and N(3) atoms and to be strongly coupled with the N(3)-H bending mode. This line can be used as an indicator for state of N(3)-H...protein interaction. The 1584-cm-1 mode, which is known to be resonance-enhanced upon excitation near the 370-nm absorption band, was accompanied by the displacement of the N(5) atom in particular. The 1355-cm-1 mode was most strongly resonance-enhanced by the 450-nm absorption band and involved the displacements of all carbon atoms of ring III. Both lines can be used as structure probes for elucidating the structure of electronically excited states of isoalloxazine.     

A resonance Raman study on the structures of complexes of flavoprotein D-amino acid oxidase

Resonance Raman (RR) spectra were obtained for the purple complexes of D-amino acid oxidase (DAO) with D-lysine or N-methylalanine. RR spectra of a complex of oxidized DAO with the oxidation product of D-lysine or D-proline were also measured. The isotope shifts of the observed bands of the purple complex with D-lysine upon 13C- or 15N-substitution of lysine indicate that the ligand is delta 1-piperideine-2-carboxylate. That the band at 1671 cm-1 for the purple intermediate with N-methylalanine shifts to 1666 cm-1 in D2O solution indicates that the imino acid, N-methyl-alpha-iminopropionate, has a protonated imino group. Many bands due to a ligand in the RR spectra of the complex of oxidized DAO with an oxidation product can be observed below 1000 cm-1, but no band for the purple complex is seen in this frequency region. The band associated with the CO2-symmetric stretching mode of the product, such as delta 1-piperideine-2-carboxylate or delta 1-pyrrolidine-2-carboxylate, complexed with the oxidized DAO shifts in D2O solution. This suggests that the product imino acid interacts with the enzyme through some proton(s).     

Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein

Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.     

Isomerization of delta 1-piperideine-2-carboxylate to delta 2-piperideine-2-carboxylate on complexation with flavoprotein D-amino acid oxidase.

The 1,646 cm-1 band in a resonance Raman spectrum obtained with excitation in the charge-transfer band of the complex of oxidized D-amino acid oxidase (DAO) with the oxidation product of D-lysine catalyzed by DAO shifted to 1,617 cm-1 upon 2-13C substitution of lysine. Thus, the band is assigned to a C(2) = C(3) stretching mode of the enamine, delta 2-piperideine-2-carboxylate (En). In the enzyme-free solution, the product is preferentially in the cyclic imine form, delta 1-piperideine-2-carboxylate (Im). Thus, DAO has a higher affinity for the enamine form than for the imine form. The pH effects on the affinity of DAO for the product and on the molar absorption coefficient at 630 nm in the charge-transfer band, suggest that the enzyme-bound product is En in the neutral form at the N atom. As the value of observed rate constant between DAO and the product was constant at high product concentrations, the binding mechanism can be explained as follows; E + Im in equilibrium with EIm in equilibrium with EEN: rapid bimolecular and slow unimolecular processes. The isomerization of the imine form to the enamine form proceeds in the slow process. The low affinity of Im for DAO may be due to a steric repulsion of the hydrogen atoms of Im at C(3) in the active site. The hydrogen atoms of a substrate D-amino acid at C(3), which correspond to the C(3) hydrogens of Im, may act repulsively in the active site and the repulsive energy may induce strain or distortion of the substrate and the enzyme, accelerating the catalytic reaction.     

Role of hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase iota

Human DNA polymerase iota (Pol iota) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol iota with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol iota active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N(6) of an A or the O(6) of a G, here we provide evidence that whereas hydrogen bonding at N(6) is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O(6) is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O(6) and N7 hydrogen bonding to DNA synthesis by Pol iota, we have examined its proficiency for replicating through the (6)O-methyl guanine and 8-oxoguanine lesions, which affect the O(6) and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O(6) is an imperative. The dispensability of N(6) hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol iota with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N(6) positions of templating A.     

13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins.

Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at -9.8 p.p.m.). The data are compared with published (31)P-NMR data of enzymes closely related to TrxR. (13)C and (15)N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C(2)O, C(4)O, N(1) and N(5). The N(10) atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C(10alpha,gamma,delta) are the most affected upon binding to the apoprotein and the large downfield shift of the C(10gamma) atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data, except for the N(1) and the N(3) atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from those of the other enzymes. From this it is concluded that C138S is in the FO conformation and the others are in the FR conformation, supporting published data. In the reduced state, strong hydrogen bonding interactions are observed between C(2)O and C(4)O and the apoprotein. As revealed by the (15)N chemical shifts and the N(5)H coupling constant the N(5) and the N(10) atom are highly sp(3) hybridized. The calculation of the endocyclic angles for the N(5) and the N(10) atoms shows the angles to be approximately 109 degrees, in perfect agreement with X-ray data showing that the flavin assumes a bent conformation along the N(10)/N(5) axis of the flavin. In contrast, the N(1) is highly sp(2) hybridized and is protonated, i.e. in the neutral state. Upon reduction of the enzymes, the (13)C chemical shifts of some atoms of the ribityl side chain undergo considerable changes also indicating conformational rearrangements of the side-chain interactions with the apoproteins. The chemical shifts between native TrxR and C138S are now rather similar and differ from those of the two other mutant proteins. This strongly indicates that the former enzymes are in the FO conformation and the other two are in the FR conformation. The data are discussed briefly in the context of published NMR data obtained with a variety of flavoproteins.