The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction.

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions.

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.

Filming DNA repair at the atomic level.

Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications.

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.

Excited-State Properties of Fully Reduced Flavins in Ferredoxin-NADP+ Oxidoreductase.

The fully reduced flavin cofactor (FADred) in ferredoxin-NADP+ oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FADred in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FADred and the dynamic properties of the protein environment, we conclude that the excited state of neutral FADred can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts.

Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase.

Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+.

Early processes in heme-based CO-sensing proteins.

Carbon monoxide has been recognized relatively recently as signaling molecule, and only very few dedicated natural CO sensor proteins have been identified so far. These include in particular heme-based transcription factors: the bacterial sensor proteins CooA and RcoM. In these 6-coordinated systems, exchange between an internal protein residue and CO as a heme ligand in the sensor domain affects the properties of the DNA-binding domain. Using light to dissociate heme-ligand bonds can in principle initiate this switching process. We review the efforts to use this method to investigate early processes in ligand switching and signaling, with an emphasis on the CO-"trappingË® properties of the heme cavity. These features are unusual for most heme proteins, but common for heme-based CO sensors.

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.

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.

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.

Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives.

Flavins are highly versatile redox-active and colored cofactors in a large variety of proteins. These do include photoenzymes and photoreceptors, although the vast majority performs non-light-driven physiological functions. Nevertheless, electron transfer between flavins and specific nearby amino acid residues (in particular tyrosine, tryptophan, and presumably histidine and arginine) takes place upon excitation of flavin in many flavoproteins. For oxidized flavoproteins these reactions potentially have a photoprotective role. In this Perspective, we outline work on the characterization of early reaction intermediates not only in the relatively well-studied resting oxidized forms but also in the fully reduced and the intrinsically unstable semireduced forms, where ultrafast photooxidation of flavin was recently demonstrated. Along different lines, flavoprotein-based novel photocatalysts for biotechnological applications are presently emerging, employing both substrate photooxidation and photoreduction strategies. Deep insight into the fundamental flavin photochemical reactions may help in guiding and optimizing their development and in the exploration of novel photocatalytic approaches.

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.

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.

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.

Fluorescent iron­sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus.

Cytochrome bc1 complexes are energy-transducing enzymes and key components of respiratory electron chains. They contain Rieske 2Fe2S proteins that absorb very weakly in the visible absorption region compared to the heme cofactors of the cytochromes, but are known to yield photoproducts. Here, the photoreactions of isolated Rieske proteins from the hyperthermophilic bacterium Aquifex aeolicus are studied in two redox states using ultrafast transient fluorescence and absorption spectroscopy. We provide evidence, for the first time in iron­sulfur proteins, of very weak fluorescence of the excited state, in the oxidized as well as the reduced state. The excited states of the oxidized and reduced forms decay in 1.5 ps and 30 ps, respectively. In both cases they give rise to product states with lifetimes beyond 1 ns, reflecting photo-reduction of oxidized centers as well as photo-oxidation of reduced centers. Potential reaction partners are discussed and studied using site-directed mutagenesis. For the reduced state, a nearby disulfide bridge is suggested as an electron acceptor. The resulting photoproducts in either state may play a role in photoactivation processes.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy.

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

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.