Photo-dynamics of the BLUF domain containing soluble adenylate ...

Chemical Physics xxx (2011) xxx–xxx

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Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain A. Penzkofer a,⇑, M. Stierl b, P. Hegemann b, Suneel Kateriya c a

Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany c Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India b

a r t i c l e

i n f o

Article history: Received 11 March 2011 In final form 18 May 2011 Available online xxxx Keywords: BLUF domain Cyclase homology domain Amoeboflagellate Naegleria gruberi BLUF-type photo-cycle One-electron reduction–oxidation photocycle Photo-degradation Flavin Flavin-semiquinone Flavin-hydroquinone Flavin anion radical

a b s t r a c t The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF (blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photodynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Bluelight exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Flox) to semi-reduced flavin (FlH) occurred. Furthermore, photo-excitation of FlH caused irreversible electron transfer to fully reduced anionic flavin (FlH). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr+–Fl or Trp+–Fl radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Flox to lumichrome was observed. A model of the photo-dynamics of nPAC is developed. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Blue-light responses of biological organisms are mediated by three classes of flavin based blue-light receptors: cryptochromes with PHR (photolyase-reated) domains [1], phototropins with LOV domains [2], and regulatory proteins with BLUF domains [3] (for reviews see [4–8]). BLUF domains (sensors of blue light using FAD) are found in many microorganisms as part of sensor (input) – actuator (effector and output) proteins [9–19] or as single sensor domain proteins [17,20–31]. The photo-cycle dynamics of the single-domain BLUF proteins BlrB from Rhodobacter sphaeroides [20,21], Tll0078 (also called TePixD) from Thermosynechococcus elongates [22,23], and Slr1694 (also called PixD) from Synechocystis sp. PCC6803 [24,25] were studied extensively. Blue-light exposure of dark-adapted BLUF proteins (in receptor state) leads to a slight red-shift of the first electronic absorption band in the light-adapted state due to photoinduced hydrogen-bond restructuring, which recovers to the initial absorption behavior after light switch-off [8,17,21,26–28]. The ⇑ Corresponding author. E-mail address: [email protected] (A. Penzkofer).

fluorescence efficiency of dark-adapted BLUF domains is small and it is even smaller in the light adapted state due to photo-induced electron transfer from an adjacent tyrosine (or tryptophan) residue to the flavin cofactor [21,27,29–31]. The BLUF domain containing sensor-actuator protein AppA from the anoxyphototropic purple bacterium R. sphaeroides antirepresses photosynthesis gene expression by blue-light exposure [17]. The BLUF-EAL domain containing blue-light regulated phosphodiesterase BlrP1 protein from the enteric bacterium Klebsiellia pneumoniae [18,26] and the BLUF-PapB–EAL-PapA proteins from the purple bacterium Rhodopseudomas palustris [19] cause bluelight dependent degradation of the bacterial specific second messenger, cyclic dimeric guanosine monophosphate (c-di-GMP). The BLUF-EAL protein YcgF from Escherichia coli does not degrade c-di-GMP but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation [32]. Its reaction is temperature sensitive and may play a role as temperature sensor [33]. BLUF domain containing adenylyl (synonymously called adenylate) cyclase domain proteins (photo-activated adenylyl cyclases, PAC) cause blue-light regulated cyclic adenylyl monophosphate formation (cAMP) from adenosine triphosphate [9–16] (cylases are enzymes that catalyze chemical reactions to form cyclic

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Please cite this article in press as: A. Penzkofer et al., Chem. Phys. (2011), doi:10.1016/j.chemphys.2011.05.028

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A. Penzkofer et al. / Chemical Physics xxx (2011) xxx–xxx

compounds). cAMP is an ubiquitous second messenger in all domains of life. The photo-activated adenylyl cyclases PACa and PACb of the unicellular flagellate Euglena gracilis (euPACs) mediate blue-light induced negative phototaxis (photophobic response) [9,10]. euPACa and euPACb were heterologously expressed in Xenopus oocytes [11], HEK cells [11], and insect cells [13]. euPACa expression in neurons of Drosophila resulted in cAMP mediated neuronal reaction of adult Drosophila flies [11] and Drosophila larvae [14]. Transplantation of euPAC into neurons of the marine gastropod Aplysia led to neuron stimulation by light [12]. Photoactivated adenylyl cyclase (called BlaC in [15] and bPAC in [16]) from the soil bacterium Beggiatoa sp. PS caused light regulated cAMP formation and was used to effectively modulate cAMP levels in neurons [15,16] showing the high potential of PAC in optogenetic applications (optogenetics = optical control of cellular parameters by genetically encoded light regulated proteins) [34–37]. Detailed photo-dynamics studies on full-length BLUF domain containing sensor-actuator proteins have been carried out up to now only for the phosphodiesterase BlrP1 protein from K. pneumoniae [26]. The overall photo-cycle behavior of the BlrP1_BLUF domain alone and of the full-length BlrP1 protein was found to be the same but the detailed kinetics and the efficiency parameters were different [26]. In this paper the photo-dynamics of a recently expressed BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain (a unicellular eukaryote) is studied in detail. This photo-activated cyclase, similar to bPAC, also shows cAMP activity in oocytes and in vitro (data not shown) but a biochemical investigation is outside the scope of the present paper. N. gruberi is a widespread free-living soil and freshwater amoeboflagellate [38]. It has a unique three-stage life cycle: amoeba, flagellate, and cyst. In culture, it will grow and divide as an amoeba. Once starved, it will transform into a flagellate, developing a microtubule cytoskeleton including flagella and basal bodies. The resulting flagellate stage is transitory, and flagellates eventually return to the amoeboid stage [39–41]. Different strains of N. gruberi exist. The genome sequence of the N. gruberi NEG-M strain (ATCC 30224) [39] was determined recently [42]. It contains 15,727 protein-coding genes enabling for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production. N. gruberi NEG-M amoeboflagellates contain at least 108 cyclases. Four of them occur in combination with BLUF domains [42], i.e. they are putative photo-activated cyclases (PACs). One of these PACs, named nPAC (accession XP_002674372; JF928492) is studied here. The amino acid sequence of nPAC is shown in Fig. 1(a). A schematic of the nPAC protein is depicted in Fig. 1(b). We heterologously expressed nPAC encoding gene in E. coli and characterized the resulting protein by absorption and fluorescence spectroscopy. The absorption cross-section spectrum, the fluorescence quantum distribution and the fluorescence decay of dark-adapted nPAC (BLUF domain in receptor state) were determined. Blue-light exposure transferred nPAC to the light-adapted state (BLUF signaling state) with characteristic red-shifted absorption and quenched fluorescence. After light switch-off, nPAC returned from the lightadapted state to the dark-adapted state with absorption and fluorescence recovery. Photo-excitation in the signaling state caused a permanent protein re-conformation and subsequent low-efficient reversible reduction of Flox to flavin semiquinone (FlH). Prolonged blue-light excitation irreversibly reduced FlH to anionic flavinhydroquinone (FlH). Some photo-degradation of Flox to lumichrome (LC) was observed. Structural formulae of the involved flavins are depicted in Fig. 1c. The photo-dynamic behavior of nPAC is discussed and compared with other flavin-based blue-light photoreceptors.

2. Experimental 2.1. Sample preparation Humanized synthetic DNA encoding the photoactivated cyclase nPAC gene of N. gruberi NEG-M (GenBank accession JF928492) was synthesized by Mr. Gene GmbH, Regensburg, Germany, and was cloned in frame behind the N-terminal His6-tag epitope into a pASK43p vector (from IBA, Göttingen, Germany). E. coli strain BL21(DE3k) was used to express the nPAC gene in LB medium (Lysogeny broth) for 48 h at 18 °C, in presence of 200 lg/l AHT (anhydrotetracyclin). The resulting nPAC protein was purified using Co-NTA-resin (from Clontech, USA) in 50 mM NaH2PO4/Na2HPO4 (pH 7.5), 300 mM NaCl, 5 mM b-mercaptoethanol, 0.1 mM phenylmethylsulfonyl chloride according to the supplier’s instruction. Eluents were dialyzed two times against 200 volumes of 10 mM NaH2PO4/Na2HPO4 (pH 7.5), 10 mM NaCl, and concentrated by ultrafiltration (Amicon Ultra-15, 10000 MWCO, from Millipore, Billerica, MA, USA). For HPLC analysis of the flavin chromophores a concentrated nPAC sample was denatured at 80 °C for 2 min and the precipitate was removed by centrifuging (11,000 g, 1 min). The supernatant was filtered through a 0.45 lm syringe filter (from Millipore) and applied to a reversed phase C18 column (250 4.6 mm2, 4 lm pore size, Synergi Polar-RP, from Phenomenex Inc., Torrance, CA, USA). The flavins were separated using 100 mM ammonium formate, 100 mM formic acid, and 40 vol.% methanol at a flow-rate of 0.8 ml/min. Elution profile was monitored by absorption measurement at 365 nm. Under these conditions FAD, FMN and riboflavin elute after 12.5, 16, and 25.5 min, respectively. The ratio of the flavin constituents was calculated by the corresponding peak intensities.

2.2. Spectroscopic techniques The nPAC samples were stored at 80 °C. Measurements were carried out at room temperature. The samples were measured in a fused silica ultra-micro cell (inner size 1.5 3 5 mm3). Absorption measurements were carried out with a spectrophotometer (Cary 50 from Varian). Fluorescence emission spectra and fluorescence excitation spectra were recorded with a fluorimeter (Cary Eclipse from Varian). Fluorescence lifetime measurements of nPAC in the darkadapted state (sample kept in the dark) were performed with a mode-locked titanium-sapphire laser system (Hurricane from Spectra-Physics) and an ultrafast streak-camera (type C1587 temporal disperser with M1952 high-speed streak unit from Hamamatsu) [43]. The samples were excited at 400 nm with pulses of 3.8 ps duration. Fluorescence with wavelength >475 nm passed to the streak camera. Fluorescence lifetime measurements of nPAC in the signaling state were carried out by transverse sample excitation at 455 nm with a LED light source (LEDC1 from Thorlabs) during fluorescence signal probing with the picosecond laser system. Photo-cycle studies were carried out by sample excitation at 455 nm (LED light source) with transmission and fluorescence probing perpendicular to the excitation path. Absorption spectra and fluorescence spectra were recorded at different times of light exposure and at different times after light switch-off. The temporal development of the absorption before, during, and after light exposure was probed at the position of strongest absorption change between light-adapted and dark-adapted state using an attenuated tungsten lamp in combination with a 491 nm interference filter and detection of the transmitted light with a photomultiplier tube (Valvo type PM 2254B) and a digital oscilloscope (LeCroy type 9361C).


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(a)

(b)

(c) H3C

R N

H3C

N

N

O NH

H3C

R N

H3C

N

Flox

Fl

H3C

-

FlH

R N N H

N-

O NH

H3C H3C

O

O

H3C

N

O NH

.-

FlH

H 3C

N

H 3C

N

H N

NH O

O

O

R N N H

N

O NH

O

.

R = ribityl-monophosphate (for FMN) or R = ribityl-diphosphateadenosine (for FAD)

LC

Fig. 1. (a) Amino acid sequence of BLUF domain-containing adenylate cyclase (nPAC) from Naegleria gruberi. (b) Schematic of primary structure of nPAC. The calculated molar mass is M = 43,962 g mol1. BLUF = sensors of blue-light using flavin. CHD = cyclase homology domain. (c) Structural formulae of flavin in oxidized state (Flox), anionic flavin radical (Fl, additional electron shown schematically in pyrazine ring), flavin semiquinone (FlH), anionic flavin hydroqinone (FlH), and lumichrome (LC).

The photo-dynamics and photochemistry of nPAC in the signaling state was studied by elongated sample excitation with intense blue-light (455 nm) and observing the transmission spectra and fluorescence spectra development during light exposure and after light exposure.

3. Results 3.1. Flavin composition, protein clustering, and cofactor loading The flavin cofactor composition of nPAC was analyzed by HPLC (see above). FMN with a mole-fraction of 70%, and FAD with a mole-fraction of 30% were found to be present. The transmission measurements of freshly thawed nPAC samples kept in the dark reveal a small transmission loss in the transparency region of the protein due to Rayleigh scattering. The light attenuation is composed of light absorption and light scattering. The attenuation coefficient spectrum [44], a(k) = aa(k) + asca(k), is the sum of the absorption coefficient spectrum, aa(k), and the scattering coefficient spectrum, asca(k). The wavelength dependence of the Rayleigh scattering is given by asca ðkÞ ¼ asca;0 ðk0 =kÞ4 where asca,0 is the scattering coefficient at wavelength k0 [44]. The attenuation coefficient spectrum of a fresh unexposed sample of nPAC, which was used to the determination of the cofactor loading, is shown by the solid curve in Fig. 2(a). Light scattering in the transparency region (>520 nm) is clearly seen. The

calculated Rayleigh scattering coefficient spectrum asca(k) is depicted by the dash–dotted curve (used parameters: k0 = 700 nm and asca,0 = 0.0088 cm1). The absorption coefficient spectrum, aa(k), is given by the dashed curve in Fig. 2(a). The Rayleigh scattering indicates a protein clustering (formation of nano-crystallites/ aggregates). For the nPAC sample of Fig. 2(a) an average degree of aggregation (number of protein monomers per cluster) of bm 126 is estimated using Eqs. (4), (8), and (9) from [45]. For cofactor loading calculation the cofactor concentration and the apo-protein concentration of nPAC were determined from the absorption coefficient spectrum, aa(k), of Fig. 2(a) and the known absorption cross-section spectra, ra,i(k), of FMN, FAD, Trp, and Tyr. The absorption cross-section spectra of the components FMN, FAD, Trp, and Tyr in neutral aqueous solution are shown in Fig. 2(b). The obtained total flavin number density is N0 4.28 h R R ~Þdm ~ ¼ N 0 xFMN S S ra;FMN ðm ~Þdm ~ þ xFAD 1016 cm3 (relation S0 S1 aa ðm 0 1 R r ðm~Þdm~ was used with xFMN = 0.7 the mole-fraction of S0 S1 a;FAD FMN, and xFAD = 0.3 the mole-fraction of FAD). The number density of the apo-protein, Napo, is calculated from the nPAC absorption coefficient at kpr = 270 nm using the relation aa ¼ N apo nTrp ra;Trp þ nTyr ra;Tyr Þ þ N 0 ðxFMN ra;FMN þ xFAD ra;FAD Þ with nTrp = 1 and nTyr = 11 (nTrp and nTyr are the numbers of Trp and Tyr per protein, see amino acid sequence of Fig. 1a) and the absorption cross-sections depicted in Fig. 2b (ra,Trp = 2.00 1017 cm2, ra,Tyr = 4.67 1018 cm2, ra,FMN = 1.33 1016 cm2, ra,FAD = 1.20 1016 cm2). A value of Napo 3.94 1016 cm3 is obtained. The determined number densities give a cofactor loading of jload;flavin ¼ N 0 =N apo 1:09. The


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Fig. 2. (a) Attenuation coefficient spectrum, a(k) (solid line), scattering coefficient spectrum, asca(k) (dash–dotted line), and absorption coefficient spectrum, aa(k) (dashed line), of nPACd. (b) Absorption cross-section spectra, ra(k), of FMNox (from [50]), FADox (from [46]), FADH (from [51]), Trp (from [84]), and Tyr (from [84]). (c) Absorption cross-section spectra of nPAC in initial dark-adapted state (nPACd), of lumichrome (LC) in aqueous solution at pH 8 (from [55]), and of anionic fully reduced FAD (FADH, from [54]).

number indicates a small cofactor overloading of the nPAC nanoclusters, but because of experimental inaccuracies the true cofactor loading may be nearer to the perfect value of 1. Any excess flavin does not take part in the photo-cycle dynamics. It is named photo-cycle inactive flavin Flina. The absorption cross-section spectrum of fresh unexposed nPAC (nPACd) kept in the dark is shown by the solid curve in Fig. 2c (obtained from dashed curve of Fig. 2a by division through N0). It is thought that FAD is in stretched form in the nPAC protein. Under these conditions no electron transfer occurs between the adenosine part and the isoalloxazine part of FAD [46,47] and the fluorescence behavior of FAD and FMN is similar. Therefore, in the following we do not distinguish between FMN and FAD and only speak about flavin. For all BLUF domains with known crystal structure FAD was found to be in stretched form [20,23,28,48,49]. For the small amount of non-photo-cycle active flavin Flina, a single-exponential fluorescence decay with lifetime of sF,ina 4.5 ns was found (see below) giving no indication of the presence of non-photo-cycle active coiled FAD.

Fig. 3. Fluorescence quantum distributions, EF(k), of nPAC under different conditions (see legend and main text). Fluorescence excitation wavelengths, kF,exc, and fluorescence quantum yields, /F, are listed.

4 ns with a streak speed of 10 ps per pixel and a time resolution of ca. 100 ps. The fluorescence decay is fitted by a bi-exponential function (dash–dotted curve). The fast component is attributed to non-covalently bound photo-cycle active fully oxidized flavin (Flox,act) and the slow component is attributed to photo-cycle inactive fully oxidized flavin (Flox,ina, excess flavin in nano-clusters). The fluorescence lifetime of the slow component is sF,ina = 4.5 ± 0.5 ns. The fluorescence quantum yield contribution of the slow R R component is /F Flox;ina SF;sl ðtÞdt= SF ðtÞdt /F ðnPACd Þ 0:0028.

3.2. Fluorescence behavior of unexposed nPAC The fluorescence quantum distribution, EF(k), of unexposed dark-adapted nPACd (BLUF domain in initial receptor state) is shown by the solid curve in Fig. 3. The fluorescence was excited at kF,exc = 440 nm and originates from Flox. The determined fluorescence quantum yield is /F(nPACd) 0.0047. The other curves in Fig. 3 belong to blue-light light-adapted nPAC (nPACl) and to dark-adapted nPAC after light exposure (nPACd,a). They will be discussed below. The fluorescence kinetics of a fresh dark-adapted nPAC sample is shown in Fig. 4. The solid curves are the measured fluorescence traces. The dotted curves show the system response functions. The fluorescence trace in part (a) was measured over a time range of

Fig. 4. Temporal fluorescence traces of unexposed nPACd measured with streakcamera. Dotted curves are instrumental response functions. Dash–dotted curves are bi-exponential fits. Fit functions are listed in the sub-figures. (a) Steak speed 10 ps/pixel. Instrumental response function taken from [43]. (b) Streak speed 0.33 ps/pixel. Instrumental response function taken from [85].



Assuming a true fluorescence quantum yield of /F;true ¼ 0:23 for photo-cycle inactive flavin as for FMN in aqueous solution at pH 8 [50] we estimate a fraction in photo-cycle inactive state of flavin (excess flavin) xFl;ina ¼ /F Flox;ina =/F;true 0:012. The fraction of photo-cycle active flavin is xFl,act = 1 – xFl,ina 0.988. The true fluorescence quantum yield of photo-cycle active flavin in the dark adapted state is /F;true ðFlox;act Þ ¼ /F ðnPACd Þ /F Flox;ina =xFl;act 0.0019. The fluorescence trace in part (b) of Fig. 4 was measured over a time range of about 150 ps with a streak speed of 0.33 ps per pixel and a time resolution of about sres 6 ps. The fluorescence signal is assigned to the emission of Flox,act (see below fluorescence lifetime measurement in light adapted state). It has a bi-exponential shape according to SF =SF; max ¼ x1 expðt=s1 Þ þ x2 expðt=s2 Þ (see dash– dotted fit curve). The faster component has a time constant of sF;act;f ¼ ðs21 s2res Þ1=2 = 11 ± 1 ps (s1 = 12.4 ± 1 ps) and the slower component has a lifetime of sF,act,sl = s2 = 122 ± 5 ps. The short fluorescence lifetime is near to the border of the time resolution of our detection system. For this situation the fluorescence decay time constant of the fast component may be determined by exploiting fluorescence signal ratio of faster to slower component, the combined fluorescence quantum yield, and the radiative lifetime. This approach of lifetime determination is described in the appendix. It also gives a fluorescence lifetime of sF,act,f = 11 ps and a fraction of the short fluorescence component of xF,act,f 0.86 (used experimental parameters in the calculation: /F,act,f//F,act,sl = 0.546, /F(Flox,act) = /F,act,f + /F,act,sl = 0.0019, srad,prot = 14 ns, and sF,act,sl = 122 ps).

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Fig. 5. Photo-cyclic behavior of nPAC. Sample was excited with intensity Iexc = 0.25 W cm2 at wavelength kexc = 455 nm. Recording of a spectrum takes 18 s. Given times belong to middle time of recording. (a) Development of absorption coefficient spectra during light exposure. (b) Recovery of absorption spectra after light switch-off. (c) Exposure is continued after 15 h of sample recovery in the dark.

3.3. Photo-dynamics 3.3.1. Experimental observations The photo-dynamics of nPAC was studied by sample excitation at 455 nm with a light emitting diode. The development of sample absorption and sample fluorescence during and after light exposure was studied. Fig. 5 shows the development of absorption coefficient spectra (Rayleigh scattering contribution is subtracted). In part (a) the sample was excited with an intensity of Iexc = 0.25 W cm2 for a duration of 11 min. The solid curve shows the initial absorption coefficient spectrum before light exposure (nPACd). After 9 s of exposure the absorption coefficient spectrum, aa(k), of oxidized flavin Flox (flavin quinone) shifted 16 nm to the red by changing from the initial dark-adapted state nPACd to the light-adapted state nPACl (change of Flox from receptor state, Flox,r, to signalling state, Flox,s). A small amount of flavin semiquinone (FlH) was formed within the first 9 s of light exposure (absorption in range from 540 to 640 nm, absorption cross-section spectrum of FlH is shown in Fig. 2b, taken from [51]). Then for one minute of light exposure, the absorption in the semi-reduced flavin FlH absorption region increased only slightly (note logarithmic scale of ordinate). In the second minute of light exposure the amount of semi-reduced flavin FlH increased to a mole-fraction of xsq,l 0.07. In the time range from 2 to 11 min the semiquinone absorption remained rather unchanged. The absorption spectrum recovery after light switch-off is shown in Fig. 5(b). The Flox,s absorption changed already completely back to the Flox,r absorption between the time of lightswitch-off and measuring the first spectrum in the dark (24 s after switch-off). The FlH absorption relaxed on a minute timescale to an after-exposure dark-adapted equilibrium of mole-fraction xsq,d’ 0.012 of FlH content. The sample exposed in Fig. 5(a) was stored at room temperature in the dark for a period of 15 h and then it was re-excited in a second cycle. Its absorption development is shown in Fig. 5(c). The second light exposure caused the same fast conversion of Flox,r to

Flox,s as in the first excitation cycle, but the partial conversion of Flox to FlH started immediately and was nearly finished after 1 min. After the second light switch-off, both Flox and FlH recovered to the dark adapted situation with similar speed as after the first excitation (spectra not shown). In Fig. 6 the temporal development of absorption coefficients at specific wavelengths is shown (data taken from Fig. 5). In part (a) the absorption development at 490 nm is depicted where the absorption difference between the light-adapted and the darkadapted state is largest. At the moment of light switch-on the absorption rose steeply to its maximum. Then, after a delay of about 1 min, the absorption decreased slowly during exposure (conversion of Flox,act,s to FlH). After light switch-off the absorption decreased steeply towards the absorption of Flox,r in the darkadapted state. The rise in absorption at light switch-on, and the decrease in absorption at light switch-off are not resolved in Fig. 6(a). They were separately measured (see below Fig. 7). In Fig. 6(b) the absorption development at 590 nm is presented where only semi-reduced flavin FlH is absorbing. In the first excitation cycle the rise in absorption at light switch-on has a sigmoidal shape after a small initial step indicating a delay in the conversion of Flox,act,s to FlH (some protein re-conformation was necessary for reduction of Flox,act,s to FlH; a small fraction of original nPAC was already in restructured state from accidental light exposure). In the second photo-excitation cycle 15 h later (dashed-line-connected dots in Fig. 6b) the absorption at light switch-on rose (1 exp)-shaped. This behavior indicates that the first photo-excitation caused some permanent protein re-conformation at the flavin binding pocket. With prolonged exposure time the absorption at 590 nm slightly decreased by permanent conversion of semi-reduced flavin to fully reduced anionic flavin hydroquinone FlH. It is likely that flavin hydroquinone is present in its anion form since the equilibrium between FlH2 and FlH is at pKa = 6.7 [52,53]. The absorption cross-section spectrum of anionic fully reduced flavin is included in Fig. 2(c) (dash–dotted curve, from [54]). Both the amount of Flox,act,s and FlH decreased with


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Fig. 6. Absorption spectra development during and after sample exposure at fixed probe wavelengths. Data are taken from Fig. 5. (a) kpr = 490 nm (absorption development of Flox). (b) kpr = 590 nm (absorption development of FlH). Lineconnected circles belong to initially unexposed sample. Dashed-line connected dots belong to recovered sample (stored for 15 h in dark after 11 min of exposure). (c) kpr = 340 nm (lumichrome has highest absorption cross-section), and kpr = 312 nm (FlH has highest absorption cross-section).

Fig. 7. Absorption development at fixed probe wavelength of kpr = 491 nm. Pulsed sample excitation at kexc = 455 nm with intensity Iexc = 0.25 W cm2 (a) and 0.005 W cm2 (b). The transmitted probe light was detected with a photomultiplier tube.

exposure time by the irreversible conversion of FlH to FlH (equilibrium ratio [FlH]/[Flox,act,s] remained constant). After light-switchoff the absorption coefficient at 590 nm recovered exponentially to a constant value determined by the new after-exposure darkadapted FlH equilibrium content. A single-exponential fit to the

absorption decay after light switch-off gives a time constant of ssq,reox = 87 ± 7 s for the re-oxidation of FlH to Flox,act,r. The line-connected circles in Fig. 6(c) show the absorption development at 340 nm where lumichrome has a stronger absorption cross-section than Flox,r (absorption cross-section spectrum of lumichrome is shown in Fig. 2c, from [55]). At light switch-on the absorption reduced because at this wavelength Flox,s is less absorbing than Flox,r. During light exposure the absorption increased slightly because of some photo-degradation of Flox to lumichrome (LC). At light switch-off the absorption increased due to recovery of Flox,act,s to Flox,act,r. The line-connected triangles in Fig. 6(c) show the absorption development at 312 nm. At this wavelength fully reduced flavin absorbs stronger than fully oxidized flavin. At light switch-on a slight reduction in absorption occurred since the formed Flox,s is slightly less absorbing than Flox,r. It followed a delayed rise in absorption with the delayed formation of FlH. The absorption still increased during light exposure where the amount of FlH already slightly decreased. This continued rise shows the conversion of FlH to FlH. At light switch-off the absorption increased slightly due to recovery of Flox,act,s to Flox,act,r. Then the absorption remained approximately constant. No re-oxidation of fully reduced flavin was observed. It might be that FlH has formed permanent ionic bonds with positively charged amino acid residues (lysine, arginine or histidine, FlHaa+ formation). The fast BLUF-type photo-cycle dynamics of Flox,act signaling state formation and recovery was separately studied by measuring the probe light transmission at kpr = 491 nm with a photomultiplier tube and an oscilloscope (see Section 2). In Fig. 7a the sample was exposed at kexc = 455 nm with Iexc = 0.25 W cm2 for a duration of texp = 103 s. The PMT signal reached quickly its saturation value (all nPAC molecules transferred to signaling state) and then a slight signal reduction occurred (conversion of Flox to FlH). After light switch-off the PMT signal recovered with a time constant of ss,rec 9.3 s. The original voltage was not fully reached due to some photomultiplier gain saturation drift. In Fig. 7(b) the sample was exposed at kexc = 455 nm with Iexc = 0.005 W cm2 for a duration of texp = 155 s. At this lower excitation intensity not all molecules were transferred to the signaling state (population saturation intensity, Isat, is comparable to used excitation intensity, see below). The rise of the PMT signal will be used below to determine the quantum efficiency of signaling-state formation. After light switch-off the sample recovered to the dark-adapted state with a time constant of ss,rec 11.4 s. The fluorescence quantum distribution, EF(k), of nPACl after 1 min of light exposure at kexc = 455 nm with Iexc = 0.25 W cm2 is shown by the dashed curve in Fig. 3. This fluorescence spectrum is thought to be determined by photo-cycle inactive flavin (Flox,ina, see time-resolved fluorescence signal behavior below). The dash– dotted curve (fluorescence excitation at kF,exc = 440 nm) and triple-dotted curve (fluorescence excitation at kF,exc = 340 nm) were measured after light-adapted state recovery (sample excitation for 11 min at kexc = 455 nm with intensity Iexc = 0.25 W cm2). The increased fluorescence for kF,exc = 440 nm is attributed to some Flox,act release to Flox,ina. The short wavelength fluorescence contribution in the case of kF,exc = 340 nm is thought to be due to lumichrome emission, which was formed by photo-degradation. In time-resolved fluorescence signal measurements during sample exposure (nPAC in light-adapted state) only the long fluorescence contribution (sF 4.5 ns) attributed to Flox,ina remained at streak speed of 10 ps per pixel (traces not shown). At streak speed of 1 ps per pixel no fluorescence signal could be resolved above the noise level (traces not shown). The bi-exponential fluorescence signal of Flox,act which was found for nPAC in the dark adapted state was no longer present. The disappearance of the fluorescence of Flox,act in the light-adapted state indicates that



the singlet excited state of Flox,act in the signaling state has a fluorescence lifetime of sF,act,s < 1 ps. It is thought that in the signaling state there occurs a very efficient reductive electron transfer from Tyr or Trp to Flox; act; s quenching the fluorescence with subsequent non-radiative Tyr+ or Trp+–Flox,act,s charge recombination. The disappearance of the fast fluorescence component also shows that Flox,ina has no fast fluorescence contribution (no detectable presence of coiled FAD). 3.3.2. Parameter extraction The presented light dependent temporal absorption behavior is applied in the following to extract dynamics parameters. Obtained parameters are collected in Table 1. 3.3.2.1. Quantum yield of signaling state formation. The quantum efficiency of BLUF domain signaling state formation by blue-light exposure, /s, is estimated from the change of photomultiplier signal at the onsets of light exposure in Fig. 7b. /s is given by the ratio of the length-integrated number density of flavin molecules converted to the signaling state, DNs, to the number density of absorbed excitation photons by nPACd, Dnph;abs;nPACd , in a time interval, dt = t1 t0, short compared to the signaling state recovery time and the signaling state saturation time at the switch-on position of light, i.e.

/s ¼

DN s : Dnph;abs;nPACd

ð1Þ

The length integrated number density of flavin molecules converted from the receptor state to the signaling state in the time interval dt is approximately given by

DNs ¼ N0 ‘

Parameter

Value

Chromophore composition xFMN 0.7 xFAD 0.3

Comments HPLC analysis HPLC analysis

Behavior of initial dark-adapted state (nPACd) /F(Flox) 0.0047 /F(Flox,act) 0.0019 /F(Flox,ina) 0.0028 sET,r (ps) 11 ± 1 sCR,r (ps) 122 ± 5 sF,ina (ns) 4.5 ± 0.5

Increases with storage time Fig. 4b, sET,r = sF,act,f Fig. 4b, sCR,r = sF,act,sl Fig. 4a

Dark-adapted photo-cycle behavior /s 0.59 ± 0.05 ss,rec (s) 10.5 ± 1.5 ss,form (ps) 122 ± 5 sET,s (ps)