Neutral Semiquinone Research Articles

A search for radical intermediates in the photocycle of LOV domains.

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state (3)FMNH(+), the radical anion (2)FMN˙(-), or the neutral semiquinone radical (2)FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 μs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5-10.0 but increases on addition of β-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair.

Direct determination of resonance energy transfer in photolyase: structural alignment for the functional state.

Photoantenna is essential to energy transduction in photoinduced biological machinery. A photoenzyme, photolyase, has a light-harvesting pigment of methenyltetrahydrofolate (MTHF) that transfers its excitation energy to the catalytic flavin cofactor FADH¯ to enhance DNA-repair efficiency. Here we report our systematic characterization and direct determination of the ultrafast dynamics of resonance energy transfer from excited MTHF to three flavin redox states in E. coli photolyase by capturing the intermediates formed through the energy transfer and thus excluding the electron-transfer quenching pathway. We observed 170 ps for excitation energy transferring to the fully reduced hydroquinone FADH¯, 20 ps to the fully oxidized FAD, and 18 ps to the neutral semiquinone FADH•, and the corresponding orientation factors (κ2) were determined to be 2.84, 1.53 and 1.26, respectively, perfectly matching with our calculated theoretical values. Thus, under physiological conditions and over the course of evolution, photolyase has adopted the optimized orientation of its photopigment to efficiently convert solar energy for repair of damaged DNA.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P½ (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.

Role of the -PEWY-glutamate in catalysis at the Qo-site of the Cyt bc1 complex

We re-examine the pH dependence of partial processes of ubihydroquinone (QH2) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6–8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Qo-site by semiquinone (SQ) was similar in the wildtype and the Glu→Trp mutant. Since heme bL is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <103s−1 for reduction of heme bL by SQ from the domain distal from heme bL, a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q•− as electron donor to heme bL, then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H+ transfer from QH•, and delivery of the H+ to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme bL by allowing Q•− to move closer to the heme, accounts well for the observations.

The electronic structure of the neutral isoalloxazine semiquinone within Anabaena flavodoxin: New insights from HYSCORE experiments

A complete study of Anabaena flavodoxin in the neutral semiquinone state by means of the EPR pulse technique HYSCORE is here presented. The results provide new information about the hyperfine interactions of the unpaired electronic spin and the nuclei in the isoalloxazine ring. This allows a better knowledge of the electronic structure of the neutral flavin radical within the protein. Combination of these results with other previously obtained by using other EPR related techniques allowed producing a very precise mapping of the flavin spin distribution in the neutral semiquinone state. This information can be very useful for determining the relationship between the electronic structure and mechanisms in flavoproteins. An experimental protocol for measuring the electronic structure details available to date is suggested.

Neutral Histidine and Photoinduced Electron Transfer in DNA Photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.

Triplets, radical cations and neutral semiquinone radicals of lumiflavin and riboflavin: An overhaul of previous pump–probe data and new multichannel absolute absorption spectra

Though long-lived transient photoproducts of flavins have received much attention over the last five decades, their spectroscopic characterization remains rudimentary and altogether inconclusive. Lumiflavin and riboflavin have therefore been reexamined, using nanosecond laser photolysis and multichannel detection over a wide spectral range (230–900 nm); differential absorption spectra of flavin solutions in aqueous buffer at neutral pH have been recorded; radical cations and semiquinone radicals were generated by quenching the triplets with C (NO 2) 4 (electron acceptor) and NO 2 - (electron donor), respectively. The problem of estimating the extent of ground state depletion after a single flash has been solved by developing a new strategy that provides reliable lower and upper bounds for the depletion (and for the molar absorption coefficients of the transient species of interest). The spectra of the neutral radicals and triplets of the two flavins are almost identical, but those of the two cations are not.

New Insights into the Ultrafast Photophysics of Oxidized and Reduced FAD in Solution

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (∼5.4 ps), the open (extended) conformation of the monomer (∼2.8 ns), and the dimer (∼27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(•).

Structural and pH Dependence of Excited State PCET Reactions Involving Reductive Quenching of the MLCT Excited State of [RuII(bpy)2(bpz)]2+ by Hydroquinones

The proton-coupled electron transfer (PCET) reaction between the bpz-based photoexcited (3)MLCT state of [Ru(II)(bpy)(2)(bpz)](2+) (bpy = 2,2'-bipyridine, bpz = 2,2'-bipyrazine) and a series of substituted hydroquinones (H(2)Q) has been studied by transient absorption (TA) and time-resolved electron paramagnetic resonance (TREPR) spectroscopy at X-band. When the reaction is carried out in a CH(3)CN/H(2)O mixed solvent system with unsubstituted hydroquinone, the neutral semiquinone radical (4a) and its conjugate base, the semiquinone radical anion (4b), are both observed. Variation of the acid strength in the solvent mixture allows the acid/base dependence of the PCET reaction to be investigated. In solutions with very low acid concentrations, TREPR spectra exclusively derived from radical anion 4b are observed, while at very high acid concentrations, the spectrum is assigned to the protonated structure 4a. At intermediate acid concentrations, either a superposition of spectra is observed (slow exchange between 4a and 4b) or substantial broadening in the TREPR spectrum is observed (fast exchange between 4a and 4b). Variation of substituents on the H(2)Q ring substantially alter this acid/base dependence and provide a means to investigate electronic effects on both the ET and PT components of the PCET process. The TA results suggest a change in mechanism from PCET to direct ET quenching in strongly basic solutions and with electron withdrawing groups on the H(2)Q ring system. Changing the ligand on the Ru complex also alters the acid/base dependence of the TREPR spectra through a series of complex equilibria between protonated and deprotonated hydroquinone radicals and anions. The relative intensities of the signals from radical 4a versus 4b can be rationalized quantitatively in terms of these equilibria and the relevant pK(a) values. An observed equilibrium deuterium isotope effect supports the conclusion that the post-PCET HQ(•)/Q(•-) equilibrium is the most important in determining the 4a/4b ratio at early delay times.

Improved Density Functional Description of the Electrochemistry and Structure−Property Descriptors of Substituted Flavins

The energetics of electrochemical changes have been investigated for several substituted flavins with the M06-L density functional. The reduction potentials for one- and two-electron reductions of these molecules have been determined and the results are consistent with experimental findings with a mean unsigned error of only 42 mV. It is especially noteworthy that the M06-L density functional makes a significant difference in the computed free energy of the first reduction of lumiflavin, which produces a neutral semiquinone. We also investigate the effects of flavin ring substituents on the geometries, charge distributions, reduction potentials, pK(a)'s, ionization potentials, electron affinities, hardnesses, softnesses, electrophilic powers, and nucleophilicities.

Dynamics of flavin semiquinone protolysis in l-α-hydroxyacid-oxidizing flavoenzymes - a study using nanosecond laser flash photolysis

The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously (El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol 66, 591-595) using time-resolved spectroscopy. In the present work, we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b(2) FDH, two homologous flavoproteins belonging to the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes. For both proteins, pulsed laser irradiation at 355 nm of the reduced enzyme generated initially the neutral semiquinone, which has rarely been observed previously for these enzymes, and hydrated electron. The radical evolved with time to the anionic semiquinone that is known to be stabilized by these enzymes at physiological pH. The deprotonation kinetics were biphasic, with durations of 1-5 micros and tens of microseconds, respectively. The fast phase rate increased with pH and Tris buffer concentration. However, this increase was about 10-fold less pronounced than that reported for the neutral semiquinone free in aqueous solution. pK(a) values close to that of the free flavin semiquinone were obtained from the transient protolytic equilibrium at the end of the fast phase. The second slow deprotonation phase may reflect a conformational relaxation in the flavoprotein, from the fully reduced to the semiquinone state. The anionic semiquinone is known to be an intermediate in the flavocytochrome b(2) catalytic cycle. In light of published kinetic studies, our results indicate that deprotonation of the flavin radical is not rate-limiting for the intramolecular electron transfer processes in this protein.

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH(*) that was formed in approximately 1 ps and recombines in 10 ps and is tentatively assigned to a FADH(*)-W8(*) radical pair. The second deactivation branch forms FADH(*) in 8 ps and evolves to FAD(*-) in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP(+) oxidoreductase (FNR) is known to provide NADPH for CO(2) assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR(S)) similar to the one found in plant plastids and a large one (FNR(L)) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR(L) exhibits a higher NADP(+)/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR(L,) named FNR(L)-PC. The enzymatic activities of FNR(L)-PC were compared with those of FNR(S). During NADPH oxidation, FNR(L)-PC exhibits a 30% decrease in the Michaelis constant K(m)((NADPH)), and a 70% increase in K(m)((ferredoxin)), which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP(+) reduction, the FNR(L)-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP(+). The increase in K(m)((ferredoxin)) and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR(L)-PC. Both isoforms are capable of catalyzing the NADP(+) reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.

Ultrafast Excited State Dynamics of 5,6-Dihydroxyindole, A Key Eumelanin Building Block: Nonradiative Decay Mechanism

As part of a program designed to elucidate the excited state properties of key eumelanin building blocks, we report herein a study of 5,6-dihydroxyindole (DHI) in phosphate buffer at pH 3 and pH 7 using femtosecond transient absorption spectroscopy. The transient absorption changes following excitation at 266 nm were used to directly monitor relaxation of the excited states. It was found that the initially generated excited state of DHI, exhibiting two main absorption bands at approximately 450 and approximately 550 nm, decays with a time constant of 5-10 ps to the equilibrated singlet excited state characterized by a very similar spectrum. This latter state then decays to the ground state and the triplet state with a characteristic time of approximately 140-180 ps. Concomitant with the singlet excited state decay of DHI, spectral features characteristic of the DHI cation radical (band at approximately 575 nm) and the triplet state (band at 440-450 nm) are detected. These species do not decay further since geminate recombination of the solvated electron and the DHI radical cation, as well as deprotonation of the cation to form the neutral semiquinone radical, occur on a time scale longer than that covered by the present experiments. These results offer novel insights into the mechanisms of nonradiative decay of eumelanin building blocks of possible relevance to the putative photoprotective and phototoxic roles of these biopolymers.

Redox Properties of the Prosthetic Groups of Na+-Translocating NADH:Quinone Oxidoreductase. 2. Study of the Enzyme by Optical Spectroscopy

Redox titration of the electronic spectra of the prosthetic groups of the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from Vibrio harveyi at different pH values showed five redox transitions corresponding to the four flavin cofactors of the enzyme and one additional transition reflecting oxidoreduction of the [2Fe-2S] cluster. The pH dependence of the measured midpoint redox potentials showed that the two-electron reduction of the FAD located in the NqrF subunit was coupled with the uptake of only one H(+). The one-electron reduction of neutral semiquinone of riboflavin and the formation of anion flavosemiquinone from the oxidized FMN bound to the NqrB subunit were not coupled to any proton uptake. The two sequential one-electron reductions of the FMN residue bound to the NqrC subunit showed pH-independent formation of anion radical in the first step and the formation of fully reduced flavin coupled to the uptake of one H(+) in the second step. All four flavins stayed in the anionic form in the fully reduced enzyme. None of the six redox transitions in Na(+)-NQR showed dependence of its midpoint redox potential on the concentration of sodium ions. A model of the sequence of electron transfer steps in the enzyme is suggested.

Spectroscopic characterization of a (6-4) photolyase from the green alga Ostreococcus tauri

The cofactor content of OtCPF1, a (6-4) photolyase isolated from the green marine alga Ostreococcus tauri, was characterized by steady-state absorption and fluorescence spectroscopy. The heterologously expressed, GST-fused, purified protein (MW: 89 kDa) is non-covalently bound to flavin adenine dinucleotide (FAD), with a flavin to apoprotein molecular ratio of 64%. No light-harvesting chromophore was found in this protein. In freshly purified OtCPF1, FAD is present in three different redox states: the fully oxidized form (FAD ox, 82%), the neutral semiquinone (FADH , 14%) and the fully reduced anion (FADH −, 4%). Keeping the sample in the dark, at 5 °C, yields oxidation of FADH and FADH −, partial release of FAD to the solution and slow degradation of the protein. Upon steady-state blue-light irradiation of OtCPF1 at 450 nm, photoreduction processes leading to an accumulation of stable FADH and FADH − species are observed. We demonstrate that this accumulation is due to the presence of an external electron donor agent in the purification buffer. Composition changes observed under steady-state photoexcitation are interpreted in terms of photoinduced reductions of FAD ox and FADH states and competitive back reactions. Specific irradiation by red light at 620 nm shows both photoreduction of FADH to FADH − and irreversible oxidation of FADH to FAD ox. The photoinduced oxidation reaction is believed to be indirectly caused by the external donor agent present in the buffer. Photoexcitation is also shown to stabilize the binding of FAD to the protein. We suggest this effect to be due to slight changes in the protein conformation, possibly strengthening the hydrogen-bonding network surrounding FAD.

Primary Steps of the Na+-translocating NADH:Ubiquinone Oxidoreductase Catalytic Cycle Resolved by the Ultrafast Freeze-Quench Approach

The Na(+)-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR) is a component of respiratory chain of various bacteria, and it generates a redox-driven transmembrane electrochemical Na(+) potential. Primary steps of the catalytic cycle of Na(+)-NQR from Vibrio harveyi were followed by the ultrafast freeze-quench approach in combination with conventional stopped-flow technique. The obtained sequence of events includes NADH binding ( approximately 1.5 x 10(7) m(-1) s(-1)), hydride ion transfer from NADH to FAD ( approximately 3.5 x 10(3) s(-1)), and partial electron separation and formation of equivalent fractions of reduced 2Fe-2S cluster and neutral semiquinone of FAD ( approximately 0.97 x 10(3) s(-1)). In the last step, a quasi-equilibrium is approached between the two states of FAD: two-electron reduced (50%) and one-electron reduced (the other 50%) species. The latter, neutral semiquinone of FAD, shares the second electron with the 2Fe-2S center. The transient midpoint redox potentials for the cofactors obtained during the fast kinetics measurements are very different from ones achieved during equilibrium redox titration and show that the functional states of the enzyme realized during its turning over cannot be modeled by the equilibrium approach.

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.

The electrochemical oxidation of phenylenediamines

Five p-phenylenediamines were oxidized electrochemically at a glassy carbon rotating ring-disk-electrode assembly. The overall reaction comprises the transfer of two electrons per molecule. At low to intermediate potentials the rate determining step for all the five compounds is the acid dissociation of the semiquinonediimine radical cation formed in a pre-equilibrium involving the transfer of one electron. Only at higher potentials can the radical cation be oxidized in parallel to the neutral semiquinone radical to form the quinonediimines. In contrast, the neutral semiquinonediimine radicals are oxidized at the same or even a lower potential than the parent phenylenediamines. The dissociation rate constants of the radical cations are in the range of 10–300 s −1. The effect of the substituents on the oxidation potentials is as expected by their property to donate electrons to the mesomeric system. Their effect on the dissociation rates, however, seems to rather correlate with the inductive effect and might influence not only the electronic structure of the molecule itself but also the structure of the ions and the solvent around them. The semiquinonediimine radical cations are found to be slightly more acidic than their protonated parent phenylenediamine cations with pK a values somewhat below 6.