Semiquinone State Research Articles

Functional Characterization of the re-Face Loop Spanning Residues 536−541 and Its Interactions with the Cofactor in the Flavin Mononucleotide-Binding Domain of Flavocytochrome P450 from Bacillus megaterium

Flavocytochrome P450BM-3, a bacterial monooxygenase, contains a flavin mononucleotide-binding domain bearing a strong structural homology to the bacterial flavodoxin. The flavin mononucleotide (FMN) serves as the one-electron donor to the heme iron, but in contrast to the electron transfer mechanism of mammalian cytochrome P450 reductase, the FMN semiquinone state is not thermodynamically stable and appears transiently as the anionic rather than the neutral form. A unique loop region comprised of residues (536)Y-N-G-H-P-P(541), which forms a type I' reverse turn and provides several interactions with the FMN isoalloxazine ring, was targeted in this study. Nuclear magnetic resonance studies support the presence of a strong hydrogen bond between the backbone amide of Asn537 and FMN N5, the anionic ionization state of the hydroquinone, and for a change in the hybridization state of the N5 upon reduction. Replacement of Tyr536, which flanks the flavin ring, with a basic residue (histidine or arginine) did not significantly influence the redox properties of the FMN or the accumulation of the anionic semiquinone. The central residues of the type I' turn (Asn-Gly) were replaced with various combinations of glycine and alanine as a means of altering the turn and its interactions. Gly538 was found to be crucial in maintaining the type I' turn conformation of the loop and the strong H-bonding interaction at N5. The functional role of the tandem Pro-Pro sequence which anchors and possible "rigidifies" the loop was investigated through alanine replacements. Despite changes in the stabilities of the oxidized and hydroquinone redox states of the FMN, none of the replacements studied significantly altered the two-electron midpoint potentials. Pro541 does contribute to some degree to the strength of the N5 interaction and the formation of the anionic semiquinone. Unlike that of the flavodoxin, it would appear that the conformation of the FMN rather than the loop changes in response to reduction in this flavoprotein.

Effect of the Insertion of a Glycine Residue into the Loop Spanning Residues 536−541 on the Semiquinone State and Redox Properties of the Flavin Mononucleotide-Binding Domain of Flavocytochrome P450BM-3 from Bacillus megaterium

Despite sharing sequence and structural similarities with other diflavin reductases such as NADPH-cytochrome P450 reductase (CPR) and nitric oxide synthase, flavocytochrome P450BM-3 displays some unique redox and electron transferring properties, including the inability to thermodynamically stabilize the neutral semiquinone (SQ) state of the flavin mononucleotide (FMN) cofactor. Rather, the anionic SQ species is only transiently formed during rapid reduction. Why is this? The absence of a conserved glycine residue and, as a consequence, the shorter and less flexible cofactor-binding loop in P450BM-3 represents a notable difference from other diflavin reductases and the structurally related flavodoxin. This difference may facilitate the formation of a strong hydrogen bond between backbone amide NH group of Asn537 and N5 of the oxidized FMN, an interaction not found in the other proteins. In the flavodoxin, the conserved glycine residue plays a crucial role in a redox-linked conformational change that contributes to the thermodynamic stabilization of the neutral SQ species of the FMN through the formation of a hydrogen bond with the N5H group of the flavin. In this study, a glycine residue was inserted after Tyr536 in the loop within the isolated FMN-binding domain as well as the diflavin reductase domain of P450BM-3, a position equivalent to Gly141 in human CPR. As a result, the insertion variant was observed to accumulate the neutral form of the FMN SQ species much like CPR. The midpoint potential for the SQ/HQ couple decreased by 68 mV, while that for the OX/SQ couple remained unchanged. (15)N NMR data provide evidence of the disruption of the hydrogen bond between the backbone amide group of Asn537 and the N5 atom in the oxidized state of the FMN. Molecular models suggest that the neutral FMN SQ could be stabilized through hydrogen bonding with the backbone carbonyl group of the inserted glycine residue in a manner similar to that of CPR and the flavodoxin. The insertion of the glycine at the same location within the diflavin domain resulted in a purified protein that retained nearly stoichiometric levels of bound FAD but tended to lose the FMN cofactor. This preparation retained one-third of the ferricyanide reductase activity but <1% of the cytochrome c reductase activity of the wild type. However, the insertion variant reconstituted with FMN regained nearly half of the wild-type cytochrome c reductase activity. These results demonstrate the importance of the unique structural characteristics of the shorter loop in P450BM-3 in establishing the unique redox properties of the FMN in this protein but not its general cytochrome reductase activity.

Interaction of Flavin Adenine Dinucleotide (FAD) with a Glassy Carbon Electrode Surface

The interaction of flavin adenine dinucleotide (FAD) with a glassy carbon electrode (GCE) surface was investigated in terms of the FAD adsorption thermodynamics and kinetics, the subsequent electroreduction mechanism, and the corresponding electron-transfer rate. The kinetics of FAD electroreduction at the GCE was found to be an adsorption-controlled process. A set of electroreduction kinetic parameters was calculated: the true number of electrons involved in the FAD reduction, n=1.76, the apparent transfer coefficient, alpha(app)=0.41, and the apparent heterogeneous electron-transfer rate constant, k(app)=1.4 s(-1). The deviation of the number of exchanged electrons from the theoretical value for the complete reduction of FAD to FADH(2) (n=2) indicates that a small portion of FAD goes to a semiquinone state during the redox process. The FAD adsorption was well described by the Langmuir adsorption isotherm. The large negative apparent Gibbs energy of adsorption (DeltaG(ads)=-39.7 +/-0.4 kJ mol(-1)) indicated a highly spontaneous and strong adsorption of FAD on the GCE. The energetics of the adsorption process was found to be independent of the electrode surface charge in the electrochemical double-layer region. The kinetics of FAD adsorption was modeled using a pseudo-first-order kinetic model.

Amino Acid Residues Interacting with Both the Bound Quinone and Coenzyme, Pyrroloquinoline Quinone, in Escherichia coli Membrane-bound Glucose Dehydrogenase

The Escherichia coli membrane-bound glucose dehydrogenase (mGDH) as the primary component of the respiratory chain possesses a tightly bound ubiquinone (UQ) flanking pyrroloquinoline quinone (PQQ) as a coenzyme. Several mutants for Asp-354, Asp-466, and Lys-493, located close to PQQ, that were constructed by site-specific mutagenesis were characterized by enzymatic, pulse radiolysis, and EPR analyses. These mutants retained almost no dehydrogenase activity or ability of PQQ reduction. CD and high pressure liquid chromatography analyses revealed that K493A, D466N, and D466E mutants showed no significant difference in molecular structure from that of the wild-type mGDH but showed remarkably reduced content of bound UQ. A radiolytically generated hydrated electron (e(aq)(-)) reacted with the bound UQ of the wild enzyme and K493R mutant to form a UQ neutral semiquinone with an absorption maximum at 420 nm. Subsequently, intramolecular electron transfer from the bound UQ semiquinone to PQQ occurred. In K493R, the rate of UQ to PQQ electron transfer is about 4-fold slower than that of the wild enzyme. With D354N and D466N mutants, on the other hand, transient species with an absorption maximum at 440 nm, a characteristic of the formation of a UQ anion radical, appeared in the reaction of e(aq)(-), although the subsequent intramolecular electron transfer was hardly affected. This indicates that D354N and D466N are prevented from protonation of the UQ semiquinone radical. Moreover, EPR spectra showed that mutations on Asp-466 or Lys-493 residues changed the semiquinone state of bound UQ. Taken together, we reported here for the first time the existence of a semiquinone radical of bound UQ in purified mGDH and the difference in protonation of ubisemiquinone radical because of mutations in two different amino acid residues, located around PQQ. Furthermore, based on the present results and the spatial arrangement around PQQ, Asp-466 and Lys-493 are suggested to interact both with the bound UQ and PQQ in mGDH.

Inner-Sphere Mechanism for Molecular Oxygen Reduction Catalyzed by Copper Amine Oxidases

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu(I), TPQ semiquinone state. A new mechanistic proposal for O2 reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects, (18)O KIEs, [kcat/KM((16,16)O2)]/[kcat/KM((16,18)O2)]. The (18)O KIE = 1.0136 +/- 0.0013 at pH 7.2 is independent of temperature from 5 degrees C to 47 degrees C and insignificantly changed to 1.0122 +/- 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible O2 binding to Cu(I) to afford a superoxide, [Cu(II)(eta(1)-O2)(-I)](+), intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu(II)(eta(1)-O2)(-II), intermediate driving the reduction of O2. The similar (18)O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.

S15/1 The QO site semiquinone state in isolated cytochrome bc1 (complex iii) from Rhodobacter capsulatus

S15/1 The QO site semiquinone state in isolated cytochrome bc1 (complex iii) from Rhodobacter capsulatus

Studies of trypanocidal (inhibitory) power of naphthoquinones: Evaluation of quantum chemical molecular descriptors for structure–activity relationships

Electronic, lipophilic and steric descriptors included in QSAR-2D and -3D are analyzed for a set of ortho- and para-naphthoquinones that have proved to be powerful oxidative agents with potent trypanocidal activities specially against Leptomonas seymouri and Trypanosoma cruzi. Electronic properties are calculated by means of semiempirical (PM3), ab initio (HF/3-21G) and density functional theory (B3LYP/6-31 + G ∗) methodologies. Three different electronic states, neutral quinones, hydroquinones and semiquinones, are studied to investigate if any one of them are statistically related with the biological activities. The best correlations were obtained at the B3LYP level of theory because it includes electronic correlation. The QSAR-2D indicates that the best trypanocidal growth inhibitors are molecules in the semiquinone electronic state, with the following properties: (a) high negative value of E HOMO, (b) high negative charge in the oxygen atoms of the carbonyl groups, (c) high positive charge in the carbon atom of one of carbonyl moieties and (d) high electronegativity ( χ). In a complementary way, the QSAR-3D indicates that the electrostatic field correlates with trypanocidal activity and the presence of bulk moieties would increase activity. The idea of comparing the three electronic states may prove to be of most importance in the general strategy to the design of new trypanocidal drugs. In fact, the experimental results showed that semiquinone is the one really statistically relevant indicating a clear connection between biochemical and theoretical aspects. Finally, we demonstrated that to be a good anti-trypanosomatid compound, the molecule must be a good electron acceptor to reach easily the essential semiquinone state. We expect that the present results motivate new experimental as well as theoretical investigations that confirm our findings.

Functional role of Coenzyme Q in the energy coupling of NADH‐CoQ oxidoreductase (Complex I): Stabilization of the semiquinone state with the application of inside‐positive membrane potential to proteoliposomes

Coenzyme Q10 (which is also designated as CoQ10, ubiquinone-10, UQ10, CoQ, UQ or simply as Q) plays an important role in energy metabolism. For NADH-Q oxidoreductase (complex I), Ohnishi and Salerno proposed a hypothesis that the proton pump is operated by the redox-driven conformational change of a Q-binding protein, and that the bound form of semiquinone (SQ) serves as its gate [FEBS Letters 579 (2005) 45-55]. This was based on the following experimental results: (i) EPR signals of the fast-relaxing SQ anion (designated as QNf(.-)) are observable only in the presence of the proton electrochemical potential (DeltamuH+); (ii) iron-sulfur cluster N2 and QNf(.-) are directly spin-coupled; and (iii) their center-to-center distance was calculated as 12angstroms, but QNf(.-) is only 5angstroms deeper than N2 perpendicularly to the membrane. After the priming reduction of Q to QNf(.-), the proton pump operates only in the steps between the semiquinone anion (QNf(.-)) and fully reduced quinone (QH2). Thus, by cycling twice for one NADH molecule, the pump transports 4H+ per 2e(-). This hypothesis predicts the following phenomena: (a) Coupled with the piericidin A sensitive NADH-DBQ or Q1 reductase reaction, DeltamuH+ would be established; (b) DeltamuH+ would enhance the SQ EPR signals; and (c) the dissipation of DeltamuH+ with the addition of an uncoupler would increase the rate of NADH oxidation and decrease the SQ signals. We reconstituted bovine heart complex I, which was prepared at Yoshikawa's laboratory, into proteoliposomes. Using this system, we succeeded in demonstrating that all of these phenomena actually took place. We believe that these results strongly support our hypothesis.

Light-dependent magnetoreception: quantum catches and opponency mechanisms of possible photosensitive molecules

Dozens of experiments on magnetosensitive, migratory birds have shown that their magnetic orientation behavior depends on the spectrum of light under which they are tested. However, it is not certain whether this is due to a direct effect on the magnetoreceptive system and which photosensitive molecules may be involved. We examined 62 experiments of light-dependent magnetoreception in three crepuscular and nocturnal migrants (48 for the European robin Erithacus rubecula, ten for the silvereye Zosterops lateralis, and four on the garden warbler Sylvia borin). For each experiment, we calculated the relative quantum catches of seven of the eight known photosensitive molecules found in the eyes of passerine birds: a short- (SW), medium- (MW) and long-wavelength (LW) cone pigment, rhodopsin, melanopsin, and cryptochrome in its fully-oxidized and semiquinone state. The following five opponency processes were also calculated: LW-SW, LW-MW, MW-SW, LW-(MW+SW), and cryptochrome-semiquinone. While the results do not clearly show which receptor system may be responsible for magnetoreception, it suggests several candidates that may inhibit the process. The two significant inhibitors of magnetoreceptive behavior were overall irradiances (from 400 to 700 nm) higher than those found at sunset and high quantum catch by the LW receptor. The results were also consistent with the hypothesis that high quantum catch by the semiquinone form of cryptochrome inhibits magnetoreception. The opponency mechanism that best separated oriented from non-oriented behavior was LW-MW, where a difference above a certain level inhibited orientation. Certain regions of experimental spectral space have been over-sampled, while large regions have not been sampled at all, including: (1) from 440 to 500 nm at all irradiance levels, (2) for wavelengths longer than 570 nm from 10(12) to 3x10(12) photons s(-1) cm(-2) and (3) for wavelengths less than 560 nm from 10(12) to 3x10(12) photons s(-1) cm(-2) and below 5x10(11) photons s(-1) cm(-2). Experiments under these conditions are needed to draw further conclusions.

Observation of an Intermediate Tryptophanyl Radical in W306F Mutant DNA Photolyase from Escherichia coli Supports Electron Hopping along the Triple Tryptophan Chain

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.

Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species

Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni2+ affinity chromatography. The aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH or dithionite or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg2+-dependent manner for which KD approximately KmIPP, suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,N-Dimethyl-2-amino-1-ethyl diphosphate (NIPP), a transition state analogue for the type I isomerase, competitively inhibits the type II enzyme, but with a much lower affinity. pH-dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UV-visible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and reoxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.

Potentiometric and Further Kinetic Characterization of the Flavin-Binding Domain ofSaccharomycescerevisiaeFlavocytochromeb2. Inhibition by Anions Binding in the Active Site

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind to the oxidized and the two-electron-reduced forms of the FDH domain, and that excess L-lactate also binds to the two-electron-reduced form. These findings point to the existence of a common or strongly overlapping binding site. A comparison of the effect of the anions on WT and R289K holo-flavocytochromes b2 indicates that invariant R289 belongs to this site. According to literature data, it must also be present in other members of the family of L-2-hydroxy acid-oxidizing enzymes.

Modulation of the Redox Properties of the Flavin Cofactor through Hydrogen-Bonding Interactions with the N(5) Atom: Role of αSer254 in the Electron-Transfer Flavoprotein from the Methylotrophic Bacterium W3A1

The functional effects of hydrogen-bonding interactions at the N(5) atom of the flavin cofactors in the oxidized state have not been well established in flavoproteins. The unique properties of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) were used to advantage in this study to evaluate this interaction. In wETF, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized state of the flavin. The strength of this hydrogen bond was systematically altered by the substitution of alphaSer254 with threonine, cysteine, or alanine by site-directed mutagenesis. The anionic semiquinone form of the flavin, which is highly stabilized both thermodynamically and kinetically in the wild-type protein, was observed to accumulate in all three mutants. However, the midpoint potential for the first couple (Eox/sq) was significantly decreased for all of the mutants, and the kinetic barrier toward the reduction of the anionic semiquinone that is observed in the wild-type wETF was effectively abolished in the alphaS254T and alphaS254C mutants. Based on the observed changes in the Kd values and associated binding energies for the flavin, the amino acid replacements destabilize both the oxidized and semiquinone states of the flavin, but to a much greater extent for the anionic semiquinone state. The Eox/sq values for the alphaSer254 mutants follow a general trend with the strength of N(5) H-bond in the oxidized state as indicated by Raman spectral analyses. These results support the conclusion that the H-bonding interaction at the N(5) plays a key role in establishing the high Eox/sq and the unusually high stability of the anionic semiquinone state in wETF.

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.

Molecular Mechanical Devices Based on Quinone−Pyrrole and Quinone−Indole Dyads: A Computational Study

A set of intramolecularly connected dyads consisting of a quinone unit and a pyrrole or indole moiety have been designed and evaluated in quantum-chemical calculations. It is shown computationally for several systems, depending on the length and attachment points of the interconnecting chains, that a reduction of the quinone to the semiquinone radical anion or quinolate dianion state leads to a reversible intramolecular reorientation from a pi-stacked to a T-stacked arrangement. In the rearranged structures, a hydrogen bond from the pyrrole or indole N-H function to the semiquinone or quinolate pi-system is created upon reduction. In some systems, hydrogen bonds to the semiquinone or quinolate oxygen atoms are partly feasible and will be preferred over T-stacking. The choice of systems has been based on recent computational observations related to photosystem I. Systems with pyrrole or indole units should provide a better basis for the envisioned molecular motor than recently proposed quinone-benzene dyads. The intramolecular interactions modify the quinone redox potentials. Electronic g-tensors have been computed for the semiquinone states. These reflect characteristically the presence and nature of hydrogen bonds to the semiquinone and represent suitable electron paramagnetic resonance spectroscopic probes for the preferred structures. Intramolecular proton transfer is possible in the dianionic state.

Spectroelectrochemical Investigation of a Flavoprotein with a Flavin-Modified Gold Electrode

A flavin-modified gold electrode was developed in order to catalyze the electrochemical oxidoreduction of flavoproteins. Surface modification was carried out by a two-step procedure. In the first step a mixed self-assembled monolayer obtained by adsorption of activated and nonactivated 3,3'-dithiopropionic acid (free acid and N-succinimidyl ester) was formed, followed by the covalent attachment of a N(10)-hexylamino-alkylated flavin derivative via an amide bond in the second step. The electrochemical properties of the flavin-modified electrode are presented and discussed. The redox potential of the attached flavin was measured at various pH values and the electron-transfer rate constant between electrode and flavin was determined as k0 = 5 s(-1) independent of pH. The flavin-modified electrode was successfully applied to the electrochemical and spectroelectrochemical investigation of the flavoprotein WrbA from Escherichia coli that shows some structural similarities to flavodoxins. It is concluded that the electron transfer "electrode --> flavin --> flavoprotein" occurs by a two-step hopping mechanism where the first step is rate determining. Kinetic details are discussed. Furthermore, it turned out that, in contrast to flavodoxins, where the semiquinone state is stabilized, WrbA rapidly takes up two electrons, directly leading to the fully reduced form. The presented electrode surface modification may generally lend itself for spectroelectrochemical investigations of flavoproteins.

Low-Temperature Interquinone Electron Transfer in Photosynthetic Reaction Centers fromRhodobacter sphaeroidesandBlastochloris viridis: Characterization of QB-States by High-Frequency Electron Paramagnetic Resonance (EPR) and Electron−Nuclear Double Resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.

Structure and spectroelectrochemical property of a ruthenium complex containing phenanthroline–quinone, and assembly of the complexes on a gold electrode

Ruthenium complexes containing pdon (pdon = 1,10-phenanthroline-5,6-dione) were synthesized. Their spectroscopic and electrochemical properties were examined. The molecular structure with [Ru(pdon)(bpy) 2](ClO 4) 2 ([ 1](ClO 4) 2) (bpy = 2,2′-bipyridyl) was determined by single crystal X-ray diffraction. The optically transparent thin-layer electrochemical measurements confirm that the quinone form of [ 1](ClO 4) 2 is reduced to the semi-quinone state in acetonitrile ( E°′ = −8 mV). Comparing the model complex, [ 1](ClO 4) 2, and metal-free pdon, the positive charge on two carbon atoms of the o-quinone group is bigger than that of metal-free pdon. The assemblies of the complexes were finally examined using ligand substitution.

Active Site Aspartate Residues Are Critical for Tryptophan Tryptophylquinone Biogenesis in Methylamine Dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.

Cis-syn thymidine dimer repair by DNA photolyase in real time.

DNA photolyase (PL) is a monomeric flavoprotein that repairs cyclobutylpyrimidine dimers (CPDs) via photoinduced electron transfer from a reduced flavin adenine dinucleotide cofactor (FADH(-)) to the bound CPD. We have used subpicosecond UV transient absorption spectroscopy to measure the electron-transfer and repair kinetics of Anacystis nidulans DNA photolyase with dimeric and pentameric oligothymidine substrates. Here we show that the electron-transfer lifetime is 32 +/- 20 ps for the pentameric substrate. Repair of the carbon-carbon double bonds (C=C) in the CPD is initiated in approximately 60 ps, and bond scission appears to be completed by 1500 ps. This suggests that the repair of the two C=C bonds proceeds sequentially and that the first bond scission has a much lower activation barrier than the second. Our experiments also suggest that the semiquinone FADH(*) cofactor is not reduced to its catalytically active FADH(-) state by substrate after repair but remains in the semiquinone state. In contrast to the longer substrate, the dinucleotide substrate produced a mixture of kinetics representing bound and unbound substrate.