Semiquinone State Research Articles

Catalytic Promiscuity of Fatty Acid Photodecarboxylase Enables Stereoselective Synthesis of Chiral α‐Tetralones

AbstractIn the field of biocatalysis, discovering novel reactivity from known enzymes has been a longstanding challenge. Fatty acid photo‐decarboxylase from Chlorella variabilis (CvFAP) has drawn considerable attention as a promising photoenzyme with potential green chemistry applications; however, its non‐natural reactivity has rarely been exploited to date. Herein we report a non‐natural reductive dehalogenation (deacetoxylation) reactivity of CvFAP inspired by its natural oxidative decarboxylation process, enabling the stereoselective synthesis of a series of chiral α‐substituted tetralones with high yields (up to 99 %) and e.r. values (up to 99 : 1). Mechanistic studies demonstrated that the native photoenzyme catalyzed the reductive dehalogenation via a novel mechanism involving oxidized state (FADox)/semiquinone state (FADsq) redox pair and an electron transfer (ET)/proton transfer (PT) process of radical termination, distinct from the previous reports. To our knowledge, this study represents a new example of CvFAP promiscuity, and thus expands the reactivity repertoire of CvFAP and highlights the versatility of CvFAP in asymmetric synthesis.

Catalytic Promiscuity of Fatty Acid Photodecarboxylase Enables Stereoselective Synthesis of Chiral α-Tetralones.

In the field of biocatalysis, discovering novel reactivity from known enzymes has been a longstanding challenge. Fatty acid photo-decarboxylase from Chlorella variabilis (CvFAP) has drawn considerable attention as a promising photoenzyme with potential green chemistry applications; however, its non-natural reactivity has rarely been exploited to date. Herein we report a non-natural reductive dehalogenation (deacetoxylation) reactivity of CvFAP inspired by its natural oxidative decarboxylation process, enabling the stereoselective synthesis of a series of chiral α-substituted tetralones with high yields (up to 99%) and e.r. values (up to 99:1). Mechanistic studies demonstrated that the native photoenzyme catalyzed the reductive dehalogenation via a novel mechanism involving oxidized state (FADox) / semiquinone state (FADsq) redox pair and an electron transfer (ET)/proton transfer (PT) process of radical termination, distinct from the previous reports. To our knowledge, this study represents a new example of CvFAP promiscuity, and thus expands the reactivity repertoire of CvFAP and highlights the versatility of CvFAP in asymmetric synthesis.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

A single hydrogen bond that tunes flavin redox reactivity and activates it for modification

A hydrogen bond from a conserved histidine tunes flavin reactivity in favour of a catalytically critical anionic semiquinone state (front). However it also favours an electronically similar anionic methide intermediate (back) leading to flavin modification.

Probing molecular interactions of semiquinone radicals at quinone reduction sites of cytochrome bc1 by X-band HYSCORE EPR spectroscopy and quantum mechanical calculations.

Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state via various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Qi) of cytochrome bc1 using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Qi to arginine) in which the SQ stability has previously been shown to markedly increase. The 14N region of the HYSCORE 2D spectrum for SQi in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome bo3. Parametrization of hyperfine and quadrupolar interactions of SQi with surrounding magnetic nuclei (1H, 14N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQi in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (1H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅−, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH−, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.

Activation mechanism of Drosophila cryptochrome through an allosteric switch.

Cryptochromes are signaling proteins activated by photoexcitation of the flavin adenine dinucleotide (FAD) cofactor. Although extensive research has been performed, the mechanism for this allosteric process is still unknown. We constructed three computational models, corresponding to different redox states of the FAD cofactor in Drosophila cryptochrome (dCRY). Analyses of the dynamics trajectories reveal that the activation process occurs in the semiquinone state FAD-●, resulting from excited-state electron transfer. The Arg381-Asp410 salt bridge acts as an allosteric switch, regulated by the change in the redox state of FAD. In turn, Asp410 forms new hydrogen bonds, connecting allosteric networks of the amino-terminal and carboxyl-terminal domains initially separated in the resting state. The expansion to a global dynamic network leads to enhanced protein fluctuations, an increase in the radius of gyration, and the expulsion of the carboxyl-terminal tail. These structural features are in accord with mutations and spectroscopic experiments.

Ultrafast photoreduction dynamics of a new class of CPD photolyases.

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950ps and ~ 50ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

Effects of active site arginine residue on redox behavior of flavins in bifurcating electron transfer flavoprotein

Flavin based electron bifurcation (FBEB) is observed in bifurcating electron transfer flavoproteins (BfEtfs). Electron bifurcation couples endergonic and exergonic electron transfer reactions to exploit lower potential electron transfer reaction step therefore this is considered as a third mode of energy conservation mechanism in biological systems. We study about the bifurcating EtfAB system from Rhodoseudomonas palustris (Rpal). This is a heterodimer containing two flavin adenine dinucleotides (FADs), one in each domain. One flavin accepts two electrons from NADH and bifurcates (Bf-FAD) each electron to higher potential electron transfer FAD (ET-FAD) and lower potential ferredoxin or flavodoxin. ET-FAD likely rests in its anionic semiquinone (ASQ) state and upon accepting an electron from the Bf-FAD it becomes a hydroquinone (HQ). BF-FAD accepts a pair of electrons from NADH to form HQ and distributes the electrons to separate acceptors without accumulating a SQ intermediate. This contrasting redox behavior of the two flavins in this system in turn points a spotlight on whether the conserved active site arginine residues have different effects in the two binding sites. R273 favors the ASQ of ET-FAD, whereas R165 near the Bf-FAD appears not to, possibly due to neutralization of its positive charge by nearby C174. R273 forms a pi- pi stacking interaction with ET-FAD whereas R165 appears to form hydrogen bond interactions with Bf-FAD. We performed site directed mutagenesis to change the size of the positively charged residue by replacing with lysine, neutralize the charge by inserting alanine and make the charge tunable via modulation of pH by introducing a histidine. Amino acid substitutions were made in each of the two flavin-binding sites separately. Based on the Spectro-electrochemical redox titrations, all the variants retained the WT pattern of single electron reactivity at the ET site and pairwise reactivity of the Bf-FAD. Reduction midpoint potential titrations showed 50% less accumulation of ASQ in R273H and R273A, whereas R273K produced similar an amount similar to that observed in WT. This confirms that positive charge helps in stabilization of ASQ. Redox titrations performed at pH 7 and 8 suggest that the substituting H273 residue remains neutral at these pHs. Amino acid replacement in the bifurcating site, R165K did not affect the formation of ASQ in ET-FAD, but removal of residue 165's positive charge in R165H resulted in diminished stability prevented FAD binding in the Bf site at pH8. Therefore, R273 plays a vital role in Bf-ETF by stabilizing the ASQ of the ET-FAD. On the other hand, R165 stabilizes the Bf-FAD that is essential for electron bifurcation in RpalEtf.

Spectral deconvolution of redox species in the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii. A flavin-dependent bifurcating enzyme

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

Charge polarization imposed by the binding site facilitates enzymatic redox reactions of quinone

Quinone reduction site (Qi) of cytochrome bc1 represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Qi allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme bH and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme bH is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5–9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQi. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Qi featuring the equilibration between the anionic and neutral states of SQi as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.

Structure of the cytochrome aa3-600 heme-copper menaquinol oxidase bound to inhibitor HQNO shows TM0 is part of the quinol binding site

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

The Ubiquinol Binding Site of Cytochrome bo3 from Escherichia coli Accommodates Menaquinone and Stabilizes a Functional Menasemiquinone.

Cytochrome bo3, one of three terminal oxygen reductases in the aerobic respiratory chain of Escherichia coli, has been well characterized as a ubiquinol oxidase. The ability of cytochrome bo3 to catalyze the two-electron oxidation of ubiquinol-8 requires the enzyme to stabilize the one-electron oxidized ubisemiquinone species that is a transient intermediate in the reaction. Cytochrome bo3 has been shown recently to also utilize demethylmenaquinol-8 as a substrate that, along with menaquinol-8, replaces ubiquinol-8 when E. coli is grown under microaerobic or anaerobic conditions. In this work, we show that its steady-state turnover with 2,3-dimethyl-1,4-naphthoquinol, a water-soluble menaquinol analogue, is just as efficient as with ubiquinol-1. Using pulsed electron paramagnetic resonance spectroscopy, we demonstrate that the same residues in cytochrome bo3 that stabilize the semiquinone state of ubiquinone also stabilize the semiquinone state of menaquinone, with the hydrogen bond strengths and the distribution of unpaired spin density accommodated for the different substrate. Catalytic function with menaquinol is more tolerant of mutations at the active site than with ubiquinol. A mutation of one of the stabilizing residues (R71H in subunit I) that eliminates the ubiquinol oxidase activity of cytochrome bo3 does not abolish activity with soluble menaquinol analogues.

Distribution, Evolution, Catalytic Mechanism, and Physiological Functions of the Flavin-Based Electron-Bifurcating NADH-Dependent Reduced Ferredoxin: NADP+ Oxidoreductase.

NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) is an electron-bifurcating enzyme first discovered in the strict anaerobes Clostridium kluyveri and Moorella thermoacetica. In vivo, Nfn catalyzes the endergonic reduction of NADP+ with NADH coupled to the exergonic reduction of NADP+ with reduced ferredoxin. Most Nfn homologs consist of two subunits, although in certain species Nfn homologs are fused. In contrast to other electron-bifurcating enzymes, Nfn possess a simpler structure. Therefore, Nfn becomes a perfect model to determine the mechanism of flavin-based electron bifurcation, which is a novel energy coupling mode distributed among anaerobic bacteria and archaea. The crystal structures of Nfn from Thermotoga maritima and Pyrococcus furiosus are known, and studies have shown that the FAD molecule of the NfnB (b-FAD) is the site of electron bifurcation, and other cofactors, including a [2Fe2S] cluster, two [4Fe4S] clusters, and the FAD molecule on the NfnA subunit, contribute to electron transfer. Further, the short-lived anionic flavin semiquinone (ASQ) state of b-FAD is essential for electron bifurcation. Nfn homologs are widely distributed among microbes, including bacteria, archaea, and probably eukaryotes, most of which are anaerobes despite that certain species are facultative microbes and even aerobes. Moreover, potential evidence shows that lateral gene transfer may occur in the evolution of this enzyme. Nfn homologs present four different structural patterns, including the well-characterized NfnAB and three different kinds of fused Nfn homologs whose detailed properties have not been characterized. These findings indicate that gene fusion/fission and gene rearrangement may contribute to the evolution of this enzyme. Under physiological conditions, Nfn catalyzes the reduction of NADP+ with NADH and reduced ferredoxin, which is then used in certain NADPH-dependent reactions. Deletion of nfn in several microbes causes low growth and redox unbalance and may influence the distribution of fermentation products. It’s also noteworthy that different Nfn homologs perform different functions according to its circumstance. Physiological functions of Nfn indicate that it can be a potential tool in the metabolic engineering of industrial microorganisms, which can regulate the redox potential in vivo.

Spectroscopic, thermodynamic and computational evidence of the locations of the FADs in the nitrogen fixation-associated electron transfer flavoprotein.

Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris (RpaETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of RpaETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT RpaETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.

High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus.

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.

Five decades of research on mitochondrial NADH-quinone oxidoreductase (complex I).

NADH-quinone oxidoreductase (complex I) is the largest and most complicated enzyme complex of the mitochondrial respiratory chain. It is the entry site into the respiratory chain for most of the reducing equivalents generated during metabolism, coupling electron transfer from NADH to quinone to proton translocation, which in turn drives ATP synthesis. Dysfunction of complex I is associated with neurodegenerative diseases such as Parkinson's and Alzheimer's, and it is proposed to be involved in aging. Complex I has one non-covalently bound FMN, eight to 10 iron-sulfur clusters, and protein-associated quinone molecules as electron transport components. Electron paramagnetic resonance (EPR) has previously been the most informative technique, especially in membrane in situ analysis. The structure of complex 1 has now been resolved from a number of species, but the mechanisms by which electron transfer is coupled to transmembrane proton pumping remains unresolved. Ubiquinone-10, the terminal electron acceptor of complex I, is detectable by EPR in its one electron reduced, semiquinone (SQ) state. In the aerobic steady state of respiration the semi-ubiquinone anion has been observed and studied in detail. Two distinct protein-associated fast and slow relaxing, SQ signals have been resolved which were designated SQNf and SQNs. This review covers a five decade personal journey through the field leading to a focus on the unresolved questions of the role of the SQ radicals and their possible part in proton pumping.

Identifying exchangeable protons in the QA‐site of photosystem II

Through the use of photosystem II (PS II), plants possess the ability to convert light energy from the sun into chemical energy that may be used for cellular functions such as carbon fixation (1). PS II has been used in the creation of a solar cell that oxidizes water to oxygen at the anode and reduces oxygen to water at the cathode thus forming an oxidation‐reduction cycle that does not produce a byproduct. Generation of such a solar cell requires electrically linking PS II to a charge transporting polymer matrix at the binding site of plastoquinone B (QB), an electron carrier found within PS II (2). Understanding more about the binding site for the redox mediators found in PS II may assist in the future development of solar cells utilizing PS II directly or modeled after PS II. Quinones play a central role in many biological electron transport chains due to their ability to participate in one electron or two electron oxidation reduction reactions (3).Interestingly, the quinones that occupy the primary quinone binding site (QA) and the secondary quinone binding site (QB−−) in PS II both have the same structure; yet, QA participates in one electron transfers while QB can be fully reduced forming a quinol that is capable of diffusing away from PS II. When QA is reduced, an anionic semiquinone free radical is formed (QA−•). Their difference in redox properties has been attributed to differences in the protein environment of the two binding sites, including hydrogen bonding interactions which can redistribute electron density stabilizing the anionic semiquinone state of QA (4). Pulsed electron paramagnetic resonance (EPR) has established the presence of hydrogen bonds between an amide nitrogen from the protein backbone and an amino nitrogen from an imidazole with the carbonyl oxygens on QA−• (5;6). Two‐dimensional (2D) pulsed EPR has resolved five‐cross peaks from the interaction of nearby protons with the unpaired electron in QA−•. Three of the cross peaks have been assigned to protons endogenous to the structure of QA−• itself–to methyl protons on the ring, β‐methylene protons, and the ring proton. Two cross peaks have been attributed to the hydrogen bond protons (5). However, the assignment of determined coupling constants to particular protons are not consistent with corresponding values that were obtained using DFT calculations (4). We have performed 2D pulsed EPR of PS II samples with QA−−• in 1H2O and 2H2O buffer. These experiments have allowed us to identify unambiguously the cross‐features from exchangeable and nonexchangeable protons and to determine more precisely the hyperfine coupling constants for them. These results provide the data set more suitable for a comparison with calculated hyperfine couplings.Support or Funding InformationPulsed EPR studies were supported by DE‐FG02‐08ER15960 Grant from Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, US DOE (S.A.D.), and NCRR/NIH Grants S10‐RR15878 and S10‐RR025438 for pulsed EPR instrumentation. Continuous wave EPR studies were supported by NSF Grant CHE‐1229498.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (E° values) to individual flavins, and the E° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-E° flavin displays sequential one-electron (1-e-) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-E° flavin displayed a single two-electron (2-e-) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.