On the role of aromatic side chains in the photoactivation of BLUF domains

BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH*-W* radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electron-transfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.     

BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms

A novel FAD-binding domain, BLUF, exemplified by the N-terminus of the AppA protein from Rhodobacter sphaeroides, is present in various proteins, primarily from Bacteria. The BLUF domain is involved in sensing blue-light (and possibly redox) using FAD and is similar to the flavin-binding PAS domains and cryptochromes. The predicted secondary structure reveals that the BLUF domain is a novel FAD-binding fold.     

Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of synechocystis sp. PCC6803

The sensor of blue-light using FAD (BLUF) domain is the flavin-binding fold categorized to a new class of blue-light sensing domain found in AppA from Rhodobacter sphaeroides and PAC from Euglena gracilis, but little is known concerning the mechanism of blue-light perception. An open reading frame slr1694 in a cyanobacterium Synechocystis sp. PCC6803 encodes a protein possessing the BLUF domain. Here, a full-length Slr1694 protein retaining FAD was expressed and purified and found to be present as an oligomeric form (trimer or tetramer). Using the purified Slr1694, spectroscopic properties of Slr1694 were characterized. Slr1694 was found to show the same red-shift of flavin absorption and quenching of flavin fluorescence by illumination as those of AppA. These changes reversed in the dark although the rate of dark state regeneration was much faster in Slr1694 than AppA, indicating that Slr1694 is a blue-light receptor based on BLUF with the similar photocycle to that of AppA. The dark decay in D(2)O was nearly four times slower than in H(2)O. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was applied to examine the light-induced structure change of a chromophore and apo-protein with deuteration and universal (13)C and (15)N isotope labeling. The FTIR results indicate that light excitation induced distinct changes in the amide I modes of peptide backbone but relatively limited changes in flavin chromophore. Light excitation predominantly weakened the C(4)=O and C(2)=O bonding and strengthened the N1C10a and/or C4aN5 bonding, indicating formational changes of the isoalloxazine ring II and III of FAD but little formational change in the isoalloxazine ring I. The photocycle of the BLUF is unique in the sense that light excitation leads to the structural rearrangements of the protein moieties coupled with a minimum formational change of the chromophore.     

Adenosine diphosphate moiety does not participate in structural changes for the signaling state in the sensor of blue-light using FAD domain of AppA

A sensor of blue light using FAD (BLUF) protein is a flavin adenine dinucleotide (FAD) based new class blue-light sensory flavoprotein. The BLUF domain of AppA was reconstituted in vitro from apoprotein and flavin adenine dinucleotide, flavin adenine mononucleotide or riboflavin. The light-induced FTIR spectra of the domain reconstituted from various flavins and the 13C-labeled apoprotein showed that identical light-induced structural changes occur in both the flavin chromophore and protein for the signaling state in all of the reconstituted holoproteins. The results showed that an adenosine 5'-dinucleotide moiety is not required for signaling-state formation in a BLUF domain.     

Photoactivated adenylyl cyclase (PAC) genes in the flagellate Euglena gracilis mutant strains.

The unicellular, green flagellate wild-type Euglena gracilis(strain Z) and its colorless phototaxis-mutant strains as well as the non-photosynthetic close relative, Astasia longa, possess several genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha(M(r) 105 kDa) and two PACbeta(90 kDa) subunits. While the proteins were first believed all to be located in the paraxonemal body (PAB), confocal microscopy revealed that Astasia longa as well as some of the mutant strains do not contain a PAB. Immunofluorescence using PAC antibodies showed that the PAC proteins are also located along the total length of the flagellum at least in some of the strains. In order to determine if the genes responsible for the PAC proteins in the PAB and flagella are identical, sequences of all PAC proteins were analyzed in the Euglena and Astasia strains studied for PAC protein location. Full sequence analysis using PCR and 3' and 5' RACE indicated a substantial divergence between strains with a homology between strains of between 45 and 100%. Sequence alignment and sequence tree construction for the main functional groups (BLUF domain, which binds FAD, and adenylyl cyclase) showed that the pacalpha and the pacbeta gene products form clusters each with some of the mutants being closely related while others show a substantial degree of genetic diversity. The conclusion of these results is that there is a family of very dissimilar PAC proteins located in the PAB and the flagellum where they serve different functions in phototaxis and step-up photophobic reactions.     

Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.     

Hydrogen bond switching among flavin and amino acid side chains in the BLUF photoreceptor observed by ultrafast infrared spectroscopy

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD^•− and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD^•− to result in FADH^• on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH^• C=N stretch marker mode, with tyrosine as the likely proton donor. FADH^• is reoxidized in 67 ps (180 ps in D₂O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by ∼180° through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.     

Light induced structural changes of a full-length protein and its BLUF domain in YcgF(Blrp), a blue-light sensing protein that uses FAD (BLUF)

Blue-light sensing proteins that use FAD (BLUF) are members of a blue-light receptor family that is widely distributed among microorganisms. The Escherichia coli YcgF protein is a BLUF protein consisting of the N-terminal FAD-binding hold (BLUF domain) and the C-terminal EAL domain. The EAL domain of YcgF is predicted to have cyclic-di-GMP phosphodiesterase activity. Light-induced structural changes for the signaling state formation were studied using the light-induced Fourier transform infrared (FTIR) difference spectroscopy of both the full-length YcgF protein (YcgF-Full) and its BLUF domain (YcgF-BLUF). YcgF-Full and YcgF-BLUF showed identical UV-visible absorption spectra of flavin in the dark state and a light-induced absorption red shift for the signaling state, which relaxed to the dark state showing identical kinetics. The light-induced FTIR difference spectrum of YcgF-Full, however, was markedly different from that of YcgF-BLUF. The spectrum of YcgF-BLUF lacked most of the IR bands that were induced in the YcgF-Full spectrum. These bands were assigned to the light-induced structural changes of the protein. However, the bands for the C4=O stretching of a FAD isoalloxazine ring were induced at the same frequency with the same band intensity in the spectra for YcgF-Full and YcgF-BLUF. Furthermore, the YcgF-Full spectrum resembled that of the YcgF-BLUF when illuminated at medium-low temperatures because of the selective suppression of protein bands. The possibility that full-length-specific protein bands are predominantly ascribed to structural changes of the C-terminal EAL domain in the signaling state as a consequence of light excitation of the N-terminal BLUF domain is discussed.     

Formate dehydrogenase from Pseudomonas oxalaticus

Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species.     

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.     

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.     

Tryptophan at position 104 is involved in transforming light signal into changes of beta-sheet structure for the signaling state in the BLUF domain of AppA

AppA is a member of an FAD-based new class blue-light sensory protein known as sensor of blue light using FAD (BLUF) protein. The spectroscopic properties of an AppA BLUF domain (AppA126), in which the tryptophan residue at position 104 had been replaced with alanine (W104A), were characterized. The W104A mutant AppA126 showed a nearly normal absorption red shift in the FAD UV-visible absorption upon illumination; however, the light state relaxed to the dark state at a rate approximately 150 times faster than that of wild-type AppA126. Light-induced structural changes of FAD and apoprotein in the wild-type and mutant AppA126 were studied by means of light-induced Fourier transform infrared (FTIR) difference spectroscopy using AppA126, in which the apoprotein had been selectively labeled with 13C. The light-induced FTIR spectrum of the W104A mutant AppA126 revealed bands corresponding to a C4 = O stretch of the FAD isoalloxazine ring and structural changes of apoprotein, but with some alterations in the bands' features. Notably, however, prominent protein bands at 1,632(+)/1,619(-) cm(-1) caused by changes in the beta-sheet structure were eliminated by the mutation, indicating that Trp104 is responsible for transforming the light signal into a specific beta-sheet structure change in the apoprotein of the AppA BLUF domain in the signaling state.     

Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human endothelial NOS reductase domain

The object of this study was to clarify the mechanism of electron transfer in the human endothelial nitric oxide synthase (eNOS) reductase domain using recombinant eNOS reductase domains; the FAD/NADPH domain containing FAD- and NADPH-binding sites and the FAD/FMN domain containing FAD/NADPH-, FMN-, and a calmodulin-binding sites. In the presence of molecular oxygen or menadione, the reduced FAD/NADPH domain is oxidized via the neutral (blue) semiquinone (FADH(*)), which has a characteristic absorption peak at 520 nm. The FAD/NADPH and FAD/FMN domains have high activity for ferricyanide, but the FAD/FMN domain has low activity for cytochrome c. In the presence or absence of calcium/calmodulin (Ca(2+)/CaM), reduction of the oxidized flavins (FAD-FMN) and air-stable semiquinone (FAD-FMNH(*)) with NADPH occurred in at least two phases in the absorbance change at 457nm. In the presence of Ca(2+)/CaM, the reduction rate of both phases was significantly increased. In contrast, an absorbance change at 596nm gradually increased in two phases, but the rate of the fast phase was decreased by approximately 50% of that in the presence of Ca(2+)/CaM. The air-stable semiquinone form was rapidly reduced by NADPH, but a significant absorbance change at 520 nm was not observed. These findings indicate that the conversion of FADH(2)-FMNH(*) to FADH(*)-FMNH(2) is unfavorable. Reduction of the FAD moiety is activated by CaM, but the formation rate of the active intermediate, FADH(*)-FMNH(2) is extremely low. These events could cause a lowering of enzyme activity in the catalytic cycle.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.     

Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.     

Structure of a cyanobacterial BLUF protein, Tll0078, containing a novel FAD-binding blue light sensor domain

The sensor proteins for blue light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. As the first structure of this BLUF domain, we have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2A resolution. Five Tll0078 monomers are located around the non-crystallographic 5-fold axis to form a pentamer, and two pentamers related by 2-fold non-crystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the N-terminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed beta-sheet with two alpha-helices running parallel with it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly conserved amino acid residues in the BLUF domain. Of these, the three apparent key residues (Asn31, Asn32 and Gln50) were substituted with Ala. Mutant proteins of N31A and N32A showed a nearly normal 10nm spectral shift of the flavin upon illumination, while the Q50A mutant did not exhibit such a shift at all. On the basis of the crystal structure, we discussed a possible role of Gln50, which is structurally and functionally linked with the critical Tyr8 (FAD-Gln50-Tyr8 network), with regard to the light-induced spectral shift of the BLUF proteins.