Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC.

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be "silent" in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.

Catalytic Mechanism of Fatty Acid Photodecarboxylase: On the Detection and Stability of the Initial Carbonyloxy Radical Intermediate.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO- occurs in 300 ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOO⋅ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCB⋅), much longer-lived linear RCOO⋅ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB⋅ decarboxylation dynamics and spectral properties and show that in contrast to DCB⋅, aliphatic RCOO⋅ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, ≪300 ps RCOO⋅ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.

Molecular insights into the role of heme in the transcriptional regulatory system AppA/PpsR.

Heme has been shown to have a crucial role in the signal transduction mechanism of the facultative photoheterotrophic bacterium Rhodobacter sphaeroides. It interacts with the transcriptional regulatory complex AppA/PpsR, in which AppA and PpsR function as the antirepressor and repressor, respectively, of photosynthesis gene expression. The mechanism, however, of this interaction remains incompletely understood. In this study, we combined electron paramagnetic resonance (EPR) spectroscopy and Förster resonance energy transfer (FRET) to demonstrate the ligation of heme in PpsR with a proposed cysteine residue. We show that heme binding in AppA affects the fluorescent properties of the dark-adapted state of the protein, suggesting a less constrained flavin environment compared with the absence of heme and the light-adapted state. We performed ultrafast transient absorption measurements in order to reveal potential differences in the dynamic processes in the full-length AppA and its heme-binding domain alone. Comparison of the CO-binding dynamics demonstrates a more open heme pocket in the holo-protein, qualitatively similar to what has been observed in the CO sensor RcoM-2, and suggests a communication path between the blue-light-using flavin (BLUF) and sensing containing heme instead of cobalamin (SCHIC) domains of AppA. We have also examined quantitatively the affinity of PpsR to bind to individual DNA fragments of the puc promoter using fluorescence anisotropy assays. We conclude that oligomerization of PpsR is initially triggered by binding of one of the two DNA fragments and observe a ∼10-fold increase in the dissociation constant Kd for DNA binding upon heme binding to PpsR. Our study provides significant new insight at the molecular level on the regulatory role of heme that modulates the complex transcriptional regulation in R. sphaeroides and supports the two levels of heme signaling, via its binding to AppA and PpsR and via the sensing of gases like oxygen.

Photoswitching Behavior of Flavin-Inhibitor Complex in a Nonphotocatalytic Flavoenzyme.

An unprecedented photoswitching phenomenon of flavin-inhibitor complexes in a flavoenzyme was revealed by femtosecond transient absorption spectroscopy. The vast majority of flavoenzymes, including monomeric sarcosine oxidase (MSOX), perform non-light-driven physiological functions. Yet, the participation of flavin cofactors in photoinduced electron transfer reactions is widespread. MSOX catalyzes the oxidative demethylation of sarcosine; methylthioacetate (MTA) is a substrate analog inhibitor that forms a complex with MSOX exhibiting intense absorption bands over the whole visible range due to flavin-MTA charge transfer (CT) interactions. Here, we demonstrate that upon excitation, these CT interactions vanish during a barrierless high quantum yield reaction in ∼300 fs. The initial complex subsequently geminately re-forms in a few nanoseconds near room temperature in a thermally activated way with an activation energy of 28 kJ/mol. We attribute this hitherto undocumented process to a well-defined photoinduced isomerization of MTA in the active site, as corroborated by experiments with the heavier ligand methylselenoacetate. Photoisomerization phenomena involving CT transitions may be further explored in photocatalytic and photoswitching applications of flavoenzymes.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase.

Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F- ) and chloride (Cl- ). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl- and F- bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl- binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F- binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity.

Characterization of Light-Induced, Short-Lived Interacting Radicals in the Active Site of Flavoprotein Ferredoxin-NADP+ Oxidoreductase.

Radicals of flavin adenine dinucleotide (FAD), as well as tyrosine and tryptophan, are widely involved as key reactive intermediates during electron-transfer (ET) reactions in flavoproteins. Due to the high reactivity of these species and their corresponding short lifetime, characterization of these intermediates in functional processes of flavoproteins is usually challenging but can be achieved by ultrafast spectroscopic studies of light-activatable flavoproteins. In ferredoxin-NADP+ oxidoreductase from Bacillus subtilis (BsFNR), fluorescence of the FAD cofactor that very closely interacts with a neighboring tyrosine residue (Tyr50) is strongly quenched. Here we study short-lived photoproducts of this enzyme and its variants, with Tyr50 replaced by tryptophan or glycine. Using time-resolved fluorescence and absorption spectroscopies, we show that, upon the excitation of WT BsFNR, ultrafast ET from Tyr50 to the excited FAD cofactor occurs in ∼260 fs, an order of magnitude faster than the decay by charge recombination, facilitating the characterization of the reaction intermediates in the charge-separated state with respect to other recently studied systems. These studies are corroborated by experiments on the Y50W mutant protein, which yield photoproducts qualitatively similar to those observed in other tryptophan-bearing flavoproteins. By combining the experimental results with molecular dynamics simulations and quantum mechanics calculations, we investigate in detail the effects of protein environment and relaxations on the spectral properties of those radical intermediates and demonstrate that the spectral features of radical anionic FAD are highly sensitive to its environment, and in particular to the dynamics and nature of the counterions formed in the photoproducts. Altogether, comprehensive characterizations are provided for important radical intermediates that are generally involved in functional processes of flavoproteins.

Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus.

Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor-acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations.

Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems.

Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm-1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals.

Ultrafast Dynamics and Vibrational Relaxation in Six-Coordinate Heme Proteins Revealed by Femtosecond Stimulated Raman Spectroscopy.

Identifying the structural rearrangements during photoinduced reactions is a fundamental challenge for understanding from a microscopic perspective the dynamics underlying the functional mechanisms of heme proteins. Here, femtosecond stimulated Raman spectroscopy is applied to follow the ultrafast evolution of two different proteins, each bearing a six-coordinate heme with two amino acid axial ligands. By exploiting the sensitivity of Raman spectra to the structural configuration, we investigate the effects of photolysis and the binding of amino acid residues in cytochrome c and neuroglobin. By comparing the system response for different time delays and Raman pump resonances, we show how detailed properties of atomic motions and energy redistribution can be unveiled. In particular, we demonstrate substantially faster energy flow from the dissociated heme to the protein moiety in cytochrome c, which we assign to the presence of covalent heme-protein bonds.

Interaction of the Full-Length Heme-Based CO Sensor Protein RcoM-2 with Ligands.

The heme-based and CO-responsive RcoM transcriptional regulators from Burkholderia xenovorans are known to display an extremely high affinity for CO while being insensitive to O2. We have quantitatively characterized the heme-CO interaction in full-length RcoM-2 and compared it with the isolated heme domain RcoMH-2 to establish the origin of these characteristics. Whereas the CO binding rates are similar to those of other heme-based sensor proteins, the dissociation rates are two to three orders of magnitude lower. The latter property is tuned by the yield of CO escape from the heme pocket after disruption of the heme-CO bond, as determined by ultrafast spectroscopy. For the full-length protein this yield is ∼0.5%, and for the isolated heme domain it is even lower, associated with correspondingly faster CO rebinding kinetics, leading to Kd values of 4 and 0.25 nM, respectively. These differences imply that the presence of the DNA-binding domain influences the ligand-binding properties of the heme domain, thus abolishing the observed quasi-irreversibility of CO binding to the isolated heme domain. RcoM-2 binds target DNA with high affinity (Kd < 2 nM) when CO is bound to the heme, and the presence of DNA also influences the heme-CO rebinding kinetics. The functional implications of our findings are discussed.

Short-Lived Radical Intermediates in the Photochemistry of Glucose Oxidase.

Glucose oxidase is a flavoprotein that is relatively well-studied as a physico-chemical model system. The flavin cofactor is surrounded by several aromatic acid residues that can act as direct and indirect electron donors to photoexcited flavin. Yet, the identity of the photochemical product states is not well established. We present a detailed full spectral reinvestigation of this issue using femtosecond fluorescence and absorption spectroscopy. Based on a recent characterization of the unstable tyrosine cation radical TyrOH•+ , we now propose that the primary photoproduct involves this species, which was previously not considered. Formation of this product is followed by competing charge recombination and radical pair stabilization reactions that involve proton transfer and radical transfer to tryptophan. A minimal kinetic model is proposed, including a fraction of TyrOH.+ that is stabilized up to the tens of picoseconds timescale, suggesting a potential role of this species as intermediate in biochemical electron transfer reactions.

Identification of the TyrOH•+ Radical Cation in the Flavoenzyme TrmFO.

Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOH•+, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOH•+ is formed in ∼1 ps by electron transfer to excited flavin and decays in ∼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates.

Ultrafast photochemistry of the bc1 complex.

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c1, two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5-7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron-sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron-sulfur clusters, in a range of protein complexes.

Correction: Ultrafast photochemistry of the bc1 complex.

Correction for 'Ultrafast photochemistry of the bc1 complex' by Marten H. Vos et al., Phys. Chem. Chem. Phys., 2017, 19, 6807-6813.

Time-resolved infrared spectroscopic studies of ligand dynamics in the active site from cytochrome c oxidase.

The catalytic site of heme-copper oxidases encompasses two close-lying ligand binding sites: the heme, where oxygen is bound and reduced and the CuB atom, which acts as ligand entry and release port. Diatomic gaseous ligands with a dipole moment, such as the signaling molecules carbon monoxide (CO) and nitric oxide (NO), carry clear infrared spectroscopic signatures in the different states that allow characterization of the dynamics of ligand transfer within, into and out of the active site using time-resolved infrared spectroscopy. We review the nature and diversity of these processes that have in particular been characterized with CO as ligand and which take place on time scales ranging from femtoseconds to milliseconds. These studies have advanced our understanding of the functional ligand pathways and reactivity in enzymes and more globally represent intriguing model systems for mechanisms of ligand motion in a confined protein environment. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX.

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity.

The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 µM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 µM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 µM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and Km(ATP) values of 0.3 min(-1) and 33.6 µM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.

Dynamics of the heme-binding bacterial gas-sensing dissimilative nitrate respiration regulator (DNR) and activation barriers for ligand binding and escape.

DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼ 7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed.

Substrate interaction dynamics and oxygen control in the active site of thymidylate synthase ThyX.

Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2'-deoxythymidine-5'-monophosphate) from dUMP (2'-deoxyuridine-5'-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s-1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s-1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms.