On the Midpoint Potential of the FAD Chromophore in a BLUF-Domain Containing Photoreceptor Protein ⇑ Jos C

FEBS Letters 585 (2011) 167–172

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On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein ⇑ Jos C. Arents, Marcela Avila Perez, Johnny Hendriks, Klaas J. Hellingwerf

Molecular Microbial Physiology Group, Swammerdam Institute for Life Science, University of Amsterdam and Netherlands Institute for Systems Biology, The Netherlands article info abstract

Article history: The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional Received 16 May 2010 regulator AppA from Rhodobacter sphaeroides equals 260 mV relative to the calomel electrode. Revised 30 September 2010 Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings Accepted 18 November 2010 this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint poten- Available online 24 November 2010 tial of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which Edited by Richard Cogdell may rationalize the primary photochemistry observed in these three flavin-containing photoreceptor families. These results also imply that LOV domains, among the flavin-containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark. Keywords: LOV domain Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Cryptochrome Redox titration Site-directed mutation Reduced and oxidized FAD Flavin semiquinone

1. Introduction These three flavin-containing photoreceptor families each use a characteristically different type of primary photochemistry (for a The molecular basis by which living organisms respond to blue review see: [4,14]): Best characterized are the LOV domains, most light has long remained a mystery, to the extent that the photosen- of which bind FMN, rather than FAD, which is the physiological sory receptors involved in this response were generally referred to chromophore in the BLUF and Cry families. In the LOV domains as ‘cryptic’ for several decades. Presumably this was in part due to light absorption leads to a singlet excited state, which converts the high-energy content of blue photons, which left little room – through inter-system crossing to a triplet state on the nanosecond when varying light intensity – between detection of the biological timescale. The triplet state then decays at the microsecond time- response and infliction of irreversible damage. This situation has scale and forms a state in which the C(4) atom of the isooxallazine changed, first with the discovery of PYP and the xanthopsin family ring of the flavin forms a covalent bond with the sulfur atom of a [1–3], and subsequently with the molecular (biological) character- nearby cysteine side chain. The deformation of the flavin ring sys- ization of three different families of flavin-containing photoreceptor tem that accompanies this covalent bond formation then leads to families [4]. The first of these latter three families were the crypto- rearrangement of its surrounding amino acid side chains, amongst chromes (Cry), of which a molecular (biological) characterization which is a key glutamine, that ‘flips’ because of this. It cannot yet was provided by Cashmore and colleagues [5]. Subsequently, also be decided whether the covalent bond formation is preceded by LOV domains [6] and BLUF domains [7] were discovered and hydrogen, electron and/or proton transfer [15]. characterized [8,9]. This has led to a situation that by now of key In BLUF domains light absorption leads to reversible electron-, representatives of each of these families the molecular structure followed by proton- [16], or hydrogen- [17] transfer at the picosec- and mechanism of primary photochemistry has been resolved and ond timescale, which then causes a similar ‘glutamine flip’ as in this knowledge has been the basis for further detailed subdivision LOV domains, as was apparent not only from structural studies of the Cry [10] and LOV families [11], as well as several interesting [18] but also suggested by trans-family engineering studies be- engineering applications (see e.g. [12,13]). tween these two photoreceptor protein families [12]. The primary electron/proton donor for the flavin is the phenolic ring of a tyrosine that hydrogen bonds in the receptor state of the BLUF domain ⇑ Corresponding author. Address: Laboratory for Microbiology, Swammerdam to the flipping glutamine, but other residues may function as a Institute for Life science, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Fax: +31 20 5257056. donor as well [19]. In Cryptochromes light absorption initiates E-mail address: [email protected] (K.J. Hellingwerf). electron transfer, from a chain of tryptophan residues, to the

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.11.035 168 J.C. Arents et al. / FEBS Letters 585 (2011) 167–172 oxidized FAD that is present in the dark. This presumably leads to a cuvette, argon was flushed over the reaction mixture at approxi- long-living bi-radical state that plays a key role in crypochrome mately 1 ml/min. signaling [20]. To ensure redox equilibrium between the added dye and the fla- In all these three families the photochemically active species in voprotein during the measurements, the concentration of xanthine the receptor state (i.e. the form present in the dark prior to light oxidase was kept low enough to allow the reduction of the compo- absorption) is the oxidized form of the FAD or FMN, as is apparent nents in the cuvette take at least two hours. Midpoint potentials from the wavelength-dependence of their activation (see e.g. [21]), (Em(protein)) can be calculated from such measurements e.g. when in spite of the fact that the intracellular environment in which such the oxidized- and reduced forms of the protein to be titrated have photoreceptors reside may become quite reduced, particularly reached the same concentration, from the relation: when cells are confronted with a lack of oxygen. For two of the ox red photoreceptor families the redox midpoint potential has recently EmðproteinÞ¼EmðdyeÞþð59=nÞ: logð½dye =½dye Þ ð1Þ been reported, i.e. for LOV domains [22] and Cryptochromes [23]. In this equation Em is the redox midpoint potential (expressed Here we report the same characteristic for the BLUF domain of relative to the standard calomel electrode), and n the number of AppA from Rhodobacter sphaeroides and several variants thereof, electrons involved in the redox transition. Either safranin O (Em = obtained through site-directed mutagenesis, and these results are 289 mV), phenosafranin (Em = 252 mV) or anthraquinone-2-sul- compared with parallel measurements on selected LOV domains. fonate (Em = 225 mV) was used as the indicator dye, with 2 lM The results obtained are discussed in the light of the mechanism methylviologen as a mediator. All these dyes are subject to a one- of primary photochemistry in the three flavin-containing blue- electron redox transition. Time series of visible spectra were light photoreceptor families and are compared with the redox- recorded using an HP8453 UV/visible spectrophotometer from and photochemical characteristics of photolyase. 280 to 700 nm [29], for at least 5000 s using an integration time of 0.5 s and with a cycle time of 30 s. 2. Materials and methods 2.4. Site-directed mutagenesis 2.1. Materials The W104A-, Q63N-, Y21F- and Q63H-variants of the BLUF do- Xanthine oxidase (from buttermilk), dyes (anthraquinone-2- main of AppA were constructed using the QuickChange site-direc- sulfonate, methylviologen, phenosafranin and safranin O), nucleo- ted mutagenesis kit (Stratagene, La Jolla, CA) and pQEAppA5-125 as tides (FMN and FAD), buffers and general chemicals were bought the template DNA for the PCR reactions [19]. All mutations were from Sigma–Aldrich Co, St. Louis, MO, USA. Glucose oxidase (lyoph- confirmed through DNA-sequence analysis. ilized) was supplied by Roche Products Ltd., Hertfordshireand, UK, and Argon gas (Argon 5.0 Instrument; >99.99% pure) was provided 2.5. Calculations by Praxair (Vlaardingen, The Netherlands). Midpoint potentials were routinely calculated from the inter- 2.2. Purification of photoreceptor domains ception with the ordinate in a plot of the redox potential of the dye versus the redox potential of the protein under study. Occa-

The BLUF domain of AppA from Rb. sphaeroides (AppA5-125) and sionally, the time series of spectral data were de-convoluted with several of its variants obtained through site-directed mutagenesis global analysis, prior to calculations of the concentration of the oxi- were isolated as previously described [24]. YtvA was purified using dized and reduced form of the photoreceptor protein. This did not the procedure described in Avila Perez et al. [25]. The LOV2 domain significantly change the outcome of the calculations. Redox mid- of phototropin-1 from Avena sativa was isolated after heterologous point potentials of mediators and dyes were taken from [27]. over-expression in Escherichia coli as described elsewhere [26]. 3. Results 2.3. Redox midpoint potential determinations 3.1. Optimization of the redox-midpoint potential assay The method to measure the redox midpoint potential (all values are expressed relative to the calomel electrode) of a series of flavin- BLUF domains, as isolated and purified from their natural host, containing photoreceptor proteins was adapted from the xanthine/ as well as after heterologous (over)expression in E. coli, have their xanthine oxidase method that was developed by Massey [27,28]. flavin in the fully oxidized form [7,30]. We therefore expect the UV/visible spectra were recorded in a 3 ml quartz cuvette which BLUF domains to gradually be converted to the reduced form under could be closed with a screw cap and containing a septum to pre- our measurement conditions. As the indicator/reporter probe sev- vent oxygen inflow and a small stirrer bean to achieve mixing of its eral dyes were tested, using the selection criteria of: (i) minimal contents within a few seconds. The reaction mixture was kept at spectral overlap between the dye and the oxidized and reduced room temperature with a forced air-flow through a cooling mantel form of the photoreceptor protein, and (ii) close match of the redox that surrounded the cuvette. All solutions were pre-treated by midpoint potential of the selected dye and the particular flavopro- bubbling with argon for at least an hour prior to spectroscopic tein under study. Using these criteria, particularly phenosafranin, measurements. The standard assay buffer contained 50 mM Tris– safranin O and anthraquinone-2-sulfonate (all three used at HCl of pH = 8.0, 1 mM EDTA, 400 lM xanthine, 2 lM methylviolo- 20 lM) were found to be suitable. In addition, in all experiments gen, 20 lM of the particular dye, 10 mM glucose and 20 lM of the 2 lM methylviologen was used as a mediator between the xan- particular flavoprotein in a total volume of 2 ml. After addition thine oxidase, the flavin-containing photosensory receptor and 10 ll of glucose oxidase (from a stock solution on 10 mg/ml), the the indicator dye. reversible reduction of the flavoprotein was initiated with 10 ll The initial experiments that we carried out showed poor repro- of xanthine oxidase (freshly prepared from a stock solution of ducibility. This has most likely been caused by slow diffusion of 15 mg/ml). Glucose and glucose oxidase were then added to re- molecular oxygen into the cuvette and could neither be prevented move molecular oxygen and keep the reaction mixture anaerobic. by tighter fitting of its cap, nor by more intense flushing with Simultaneously, via needles punched through the septum of the argon. We therefore decided to add the combination of glucose J.C. Arents et al. / FEBS Letters 585 (2011) 167–172 169 plus glucose oxidase to the contents of the cuvette. The high affin- calculate the oxidized/reduced ratio of both components. Plotting ity and turnover capacity of the latter enzyme for molecular oxy- these ratio’s against each other then allows one to calculate the re- gen (200 lM and 250 U/mg; see product sheet of the enzyme) dox-midpoint potential of the flavoprotein photoreceptor from the decreases the remaining oxygen level to very low values and known midpoint potential of the indicator dye (252 mV; see may have prevented the formation of reactive oxygen species by Section 2.3). These calculations are most conveniently carried out xanthine oxidase [31]. by using the values of the intersection of the titration curve with In this configuration a number of indicator dyes were tested, either abscise or ordinate (Fig. 1C). like anthraquinone-2-sulfonate, phenosafranine, and safranine O. Registration of complete spectra (e.g. from 300 to 650 nm) at Of these dyes, particularly phenosafranine is excellently suited regular intervals, followed by global analysis of such data (see for the titration of most of the BLUF domains. For the LOV domains, e.g. [29]), also allows one to calculate the concentration of the oxi- which have a more negative redox-midpoint potential (see further dized and reduced forms of the dye and the flavoprotein under below), both safranine O and benzyl viologen can be used. By lim- study. This approach, however, did not lead to significantly differ- iting the amount of xantine oxidase to 75 lg/ml (final concentra- ent results (data not shown). To further test the redox equilibration tion) sufficiently slow reduction is achieved so that it will take between the various redox couples in the sample cuvette during a more than 2 h before the majority of the flavoprotein has been re- titration, in a subsequent experiment, after reduction of the sample duced (Fig. 1A and B). By recording absorbance changes at 521 and with xantine and xantine oxidase, the cap of the cuvette was re- 446 nm it is then possible (after correction for the contribution of moved. To test the reversibility of sample reduction with xanthine the absorbance changes at 446 nm by the phenosafranine dye) to and xanthine oxidase, the sample was exposed (through opening the cap and slow stirring) to oxygen influx into the cuvette after most of the sample had first been reduced. The contents of the sample thus were re-oxidized during a period of 500 s. Fig. 2 shows that the plot of the redox state of the dye versus that of the flavoprotein in the reduction phase practically coincides with the same plot during the re-oxidation. This validates that the measured and

calculated E0’ values represent true equilibrium midpoint potentials (compare e.g. [22]).

3.2. Redox midpoint potentials of a series of BLUF and LOV domains

Fig. 3 shows an overview of the results obtained in our assays of the redox-midpoint potential of free flavin and of a series of flavin- containing photoreceptor domains. The redox midpoint potential of the two LOV domains (i.e. YtvA from Bacillus subtilis and the LOV2 domain of phototropin-1 from A. sativa), as measured in this study (307 and 308 mV, respectively) is close to the value reported for the LOV1 domain of the phototropin from Chlamydomonas reinhardtii (290 ± 20 mV [22]). Similar agreement exists between the measured redox-midpoint potential of FAD (and FMN; data not shown) and values reported for these nucleotides in the literature [32,33]. Significantly, the value obtained for the wild type BLUF domain of AppA is intermediate between these two values, i.e. in the order of 255 to 260 mV. The same holds for several variants of the BLUF domain, obtained through site-directed mutagenesis of single amino acids, like the W104A-, Q63N- and Y21F protein. However, upon modification of a key residue in the flavin-binding pocket

Fig. 1. Redox titration of the wild type BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides. (A) Continuous absorbance record- ings at 521 nm (solid line) and 446 nm (dashed line). The reaction was initiated at t = 200 s with addition of xanthine oxidase. (B) Full visible spectral recording (from Fig. 2. Reversibility of the reduction and oxidation of the BLUF domain of AppA, 280 to 700 nm) of the color changes in the reaction mixture during the reducing with xanthine plus xanthine oxidase and with oxygen, respectively. Data were titration. Spectra were taken at 30 s intervals, but only every tenth spectrum is obtained with 20 lM of the wild type AppA5-125 domain. The reduction reaction shown. (C) Plot of the redox potential of the BLUF domain versus that of the dye, was recorded in a period of 5000 s; the oxidation reaction was initiated by from which the redox midpoint potential can be calculated according to the method opening the lid of the cuvette and took 500 s. Solid line: oxidation; dashed line: described in the Secton 2. reduction of the ﬂavoprotein. ‘dye’: phenosafranin. 170 J.C. Arents et al. / FEBS Letters 585 (2011) 167–172

Tuning of the flavin midpoint potential by the side chains of the surrounding apo-protein in the chromophore-binding pocket can be caused by: (i) p-stacking aromatic amino acids [36]; (ii) bending of the isoalloxazine-ring [37], and (iii) hydrogen bonding from and/ or protonation by amino acid residues in the flavin-binding pocket [38]. These multiple factors make it very complicated to predict the effect of – even single – amino acid substitutions on the flavin midpoint potential. In this respect it is relevant to note that Balland et al. [23] discuss the quite strong effect of (replacement of) a spe- cific asparagine which is in close proximity to the flavin in cryptochromes and photolyases, whereas in this study the Q63 mutants show only modest alteration of the flavin midpoint potential. Many more studies will be necessary to decide which of these two obser- vation is the rule and which is the exception. Fig. 3. Calculated redox-midpoint potential of FAD, a series of BLUF domains of a flavin-containing photoreceptor protein, and of a/the LOV-domain of the photore- In the receptor state of the photosensory receptor proteins the ceptors YtvA and Phototropin 1. For details: see Section 3 and 4. Bars represent latter of the three factors seems to make the most important con- standard deviations. N = number of independent assays of the particular midpoint tribution. A change in the number of hydrogen bonds to the aro- potential. Grey: BLUF domains; black: LOV domains; open bar: aqueous solution of matic ring of the flavin is expected to shift its UV/Vis [39–42]. FAD. Values have been expressed relative to the calomel electrode. ‘AppA’ refers to residues 5–125 of the N-terminal BLUF domain of the anti-transcriptional regulator Accordingly, one would also expect a correlation between the re- AppA from Rhodobacter sphaeroides. dox midpoint potential and the UV/Vis absorption maximum of the flavin-containing photoreceptors. This correlation is indeed observed among the set of LOV- and BLUF-domains studied here (see (e.g. glutamine-63) and in the Y21F/W104F double mutant [19], Fig. 4). Although several cryptochromes have their absorbance the midpoint potential of the protein-bound flavin shifts to a maximum at even shorter wavelength than 440 nm [43,44]), also value close to the midpoint potential for free flavin in aqueous longer wavelength maxima have been reported for this photore- solution. ceptor domain (e.g. [45]), and also free FAD does not show this cor- In none of these redox titrations evidence for the formation of a relation. Significantly, flavin both in aqueous solution and in the flavosemiquinone intermediate form has been obtained. Cry domain, has largely lost its vibrational fine structure, which may explain this lack of correlation. 4. Discussion An intriguing open question that remains is the relation between the intracellular redox potential and the in vivo redox state The xanthine/xanthine oxidase method [27,28] that we selected of these flavin-containing photoreceptors. The intracellular redox for the redox titrations reported in this investigation worked well, potential differs a lot between the various relevant redox couples as can be concluded from the absence of hysteresis in Fig. 2. Be- (e.g. between NAD+/NADH and NADP+/NADPH, but for this discus- sides the general problem of slow equilibration, which is inherent sion the former couple presumably is the most important, because to many redox reactions, another crucial factor in these experi- of the abundance of this nucleotide. In the eukaryotic cytoplasm ments is the exclusion of molecular oxygen. Particularly in the the redox potential of this couple most likely is higher than most negative range of redox potentials studied, flushing with ar- 300 mV ([46]. Based on this, one can easily rationalize that LOV gon did not suffice to achieve this. Addition of glucose/glucose oxi- domains in vivo will have an oxidized flavin in the receptor state. dase, however, was sufficient to solve this problem. However, for BLUF-, and particularly for CRY domains this is less Besides BLUF and LOV domains (see Section 3.2), values for the clear. Modulation of the redox state of their flavin chromophore redox-midpoint potential of Cryptochromes and photolyases are in the receptor state may therefore form part of the regulation to available from the literature. Balland et al. [23] recently reported a value of 161 mV for the oxidized to reduced transition of Arabidopsis thaliana cryptochrome 1 (note that this photoreceptor – besides the fully reduced form -also shows flavin semiquinone formation, with a midpoint potential of 153 mV relative to the calomel electrode), very close to the 181 mV reported earlier for the same protein [34]. In the same study DNA-photolyases are reported by Balland et al. to have a midpoint potential in the range of 48 to +28 mV (depending on whether or not DNA substrate is bound), which is very close to the values for the same enzyme measured by Sokolowsky et al. [35]. Accordingly, among the three flavin-containing photoreceptor families, the midpoint potential of LOV-, BLUF- and CRY-domains increases in this order, up to values close to those for free flavin, whereas photolyases have a midpoint potential that is even higher than the one of free flavin. Significantly, this is consistent with the mechanism of primary photochemistry in the three photoreceptor families, as flavin reduction is absent in LOV domains (which have the most negative midpoint potentials), is observable at the very fast time scale in BLUF domains, and is prominent in crypto- Fig. 4. Correlation between absorbance maximum and redox midpoint potential of chromes (see also Section 1). The very low midpoint potentials of flavin-containing photoreceptor domains. Legend to Fig. 4: values for the maximal flavin absorbance of the various domains were derived from UV/Vis spectra of the LOV domains may make them very robust and on this basis may purified proteins. Symbols represent: : YtvA; }: AsLOV2; N: AppA5-125Q63 N; 4: explain their very wide phylogenetic distribution, even in very AppA5-125Y21F; j: AppA5-125W104A; h: AppA5-125; d: AppA5-125Q63H; s: AppA5- extremophilic microorganisms [11]. 125Y21F W104F. J.C. Arents et al. / FEBS Letters 585 (2011) 167–172 171 which their activity is subjected (compare [47]). In this respect it is [20] Bouly, J.P. et al. (2007) Cryptochrome blue light photoreceptors are activated relevant to note that the midpoint potential of the sulfhydryl through interconversion of flavin redox states. J. Biol. Chem. 282, 9383–9391. [21] Avila-Perez, M., Hellingwerf, K.J. and Kort, R. (2006) Blue light activates the groups of AppA, which mediate the redox regulation of its interac- sigma(B)-dependent stress response of Bacillus subtilis via YtvA. J. Bacteriol. tion with PpsR, has been estimated to be 315 mV [48]. The cyto- 188, 6411–6414. plasmic redox potential may particularly have an effect in [22] Noll, G., Hauska, G., Hegemann, P., Lanzl, K., Noll, T., von Sanden-Flohe, M. and Dick, B. (2007) Redox properties of LOV domains: chemical versus prokaryotes, as many of them can thrive in the absence of oxygen, photochemical reduction, and influence on the photocycle. ChemBioChem 8, i.e. in very reducing conditions. Therefore, a lot remains to be 2256–2264. learned about the mechanism of integration of light- and redox sig- [23] Balland, V., Byrdin, M., Eker, A.P.M., Ahmad, M. and Brettel, K. (2009) What makes the difference between a cryptochrome and DNA photolyase? A nals, particularly in BLUF domains. Of particular relevance is the spectroelectrochemical comparison of the flavin redox transitions. J. Am. question whether or not light-sensitivity of flavin photoreceptors Chem. Soc. 131, 426–427. is strictly limited to receptor states with an oxidized flavin. Re- [24] Laan, W., van der Horst, M.A., van Stokkum, I.H. and Hellingwerf, K.J. (2003) Initial characterization of the primary photochemistry of AppA, a blue-light- cently, the term ‘optogenetics’ was coined to refer to studies in using flavin adenine dinucleotide-domain containing transcriptional which engineered photoreceptors are used to study a wide range antirepressor protein from Rhodobacter sphaeroides: a key role for reversible of biological problems [49,50]. We anticipate that this field will intramolecular proton transfer from the flavin adenine dinucleotide bring more light to the details of this integration [51]. Knowledge chromophore to a conserved tyrosine? Photochem. Photobiol. 78, 290–297. [25] Avila-Perez, M., Vreede, J., Tang, Y.F., Bende, O., Losi, A., Gartner, W. and to be gained in such studies may further aid engineering efforts, Hellingwerf, K. (2009) In vivo mutational analysis of YtvA from Bacillus subtilis like the transformation of BLUF domains into LOV domains and visa mechanism of light activation of the general stress response. J. Biol. Chem. versa [12], because they form the ultimate proof of proper under- 284, 24958–24964. [26] Harper, S.M., Neil, L.C. and Gardner, K.H. (2003) Structural basis of a standing these photosensory receptor systems. phototropin light switch. Science 301, 1541–1544. [27] Massey, V. (1991) A simple method for the determination of redox potentials in: Flavins and Flavoproteins 1990 (Curti, B.R.S. and Zanetti, G., Eds.), pp. 59– References 66, Walter de Gruyter & Co, Berlin. [28] Fraaije, M.W., van den Heuvel, R.H.H., van Berkel, W.J.H. and Mattevi, A. (1999) [1] Meyer, T.E. (1985) Isolation and characterization of soluble cytochromes, Covalent flavinylation is essential for efficient redox catalysis in vanillyl- ferredoxins and other chromophoric proteins from the halophilic phototrophic alcohol oxidase. J. Biol. Chem. 274, 35514–35520. bacterium Ectothiorhodospira halophila. Biochim. Biophys. Acta 806, [29] Hendriks, J. and Hellingwerf, K.J. (2009) PH dependence of the photoactive 175–183. yellow protein photocycle recovery reaction reveals a new late photocycle [2] Sprenger, W.W., Hoff, W.D., Armitage, J.P. and Hellingwerf, K.J. (1993) The intermediate with a deprotonated chromophore. J. Biol. Chem. 284, 5277– eubacterium Ectothiorhodospira halophila is negatively phototactic, with a 5288. wavelength dependence that fits the absorption spectrum of the photoactive [30] Laan, W., Bednarz, T., Heberle, J. and Hellingwerf, K.J. (2004) Chromophore yellow protein. J. Bacteriol. 175, 3096–3104. composition of a heterologously expressed BLUF-domain. Photochem. [3] Kort, R. et al. (1996) The xanthopsins: a new family of eubacterial blue-light Photobiol. Sci. 3, 1011–1016. photoreceptors. EMBO J. 15, 3209–3218. [31] Zanotto-Filho, A., Schroder, R. and Moreira, J.C. (2008) Xanthine oxidase- [4] van der Horst, M.A. and Hellingwerf, K.J. (2004) Photoreceptor proteins, ‘‘star dependent ROS production mediates vitamin A pro-oxidant effects in cultured actors of modern times’’: a review of the functional dynamics in the structure Sertoli cells. Free Radic. Res. 42, 593–601. of representative members of six different photoreceptor families. Acc. Chem. [32] Segel, H. (1986) Biochemical Calculations, 2nd ed, John Wiley and Sons Inc., Res. 37, 13–20. New York. [5] Ahmad, M. and Cashmore, A.R. (1993) HY4 gene of A. Thaliana encodes a [33] Berg, J.M., Tymoczko, J.L. and Stryer, L. (2007) Biochemistry (sixth edition), protein with characteristics of a blue-light photoreceptor. Nature 366, 162– W.H. Freeman and Company, New York. 166. [34] Lin, C.T., Robertson, D.E., Ahmad, M., Raibekas, A.A., Jorns, M.S., Dutton, P.L. and [6] Huala, E., Oeller, P.W., Liscum, E., Han, I.S., Larsen, E. and Briggs, W.R. (1997) Cashmore, A.R. (1995) Association of flavin adenine-dinucleotide with the Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Arabidopsis blue-light receptor Cry1. Science 269, 968–970. Science 278, 2120–2123. [35] Sokolowsky, K. et al. (2010) Spectroscopic and thermodynamic comparisons of [7] Masuda, S. and Bauer, C.E. (2002) AppA is a blue light photoreceptor that Escherichia coli DNA photolyase and Vibrio cholerae Cryptochrome 1. J. Phys. antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell Chem. B 114, 7121–7130. 110, 613–623. [36] Zhou, Z.M. and Swenson, R.P. (1996) The cumulative electrostatic effect of [8] Christie, J.M., Salomon, M., Nozue, K., Wada, M. and Briggs, W.R. (1999) LOV aromatic stacking interactions and the negative electrostatic environment of (light, oxygen, or voltage) domains of the blue-light photoreceptor the flavin mononucleotide binding site is a major determinant of the phototropin (nph1): binding sites for the chromophore flavin reduction potential for the flavodoxin from Desulfovibrio vulgaris mononucleotide. Proc. Natl. Acad. Sci. USA 96, 8779–8783. [Hildenborough]. Biochemistry 35, 15980–15988. [9] Gomelsky, M. and Klug, G. (2002) BLUF: a novel FAD-binding domain involved [37] Bhattacharyya, S., Stankovich, M.T., Truhlar, D.G. and Gao, J.L. (2007) in sensory transduction in microorganisms. Trends Biochem. Sci. 27, 497–500. Combined quantum mechanical and molecular mechanical simulations of [10] Lin, C. and Todo, T. (2005) The cryptochromes. Genome Biol. 6, 220. one- and two-electron reduction potentials of flavin cofactor in water, [11] Krauss, U., Minh, B.Q., Losi, A., Gartner, W., Eggert, T., von Haeseler, A. and medium-chain acyl-CoA dehydrogenase, and cholesterol oxidase. J. Phys. Jaeger, K.E. (2009) Distribution and phylogeny of light-oxygen-voltage-blue- Chem. A 111, 5729–5742. light-signaling proteins in the three kingdoms of life. J. Bacteriol. 191, 7234– [38] Chang, F.C., Bradley, L.H. and Swenson, R.P. (2001) Evaluation of the hydrogen 7242. bonding interactions and their effects on the oxidation-reduction potentials [12] Suzuki, H., Okajima, K., Ikeuchi, M. and Noguchi, T. (2008) LOV-like flavin-Cys for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin. Biochimica adduct formation by introducing a Cys residue in the BLUF domain of TePixD. Et Biophysica Acta-Bioenergetics 1504, 319–328. J. Am. Chem. Soc. 130, 12884. [39] Nozaki, D., Iwata, T., Ishikawa, T., Todo, T., Tokutomi, S. and Kandori, H. (2004) [13] Moglich, A., Ayers, R.A. and Moffat, K. (2009) Design and signaling mechanism Role of Gln1029 in the photoactivation processes of the LOV2 domain in of light-regulated histidine kinases. J. Mol. Biol. 385, 1433–1444. adiantum phytochrome3. Biochemistry 43, 8373–8379. [14] Moglich, A., Yang, X., Ayers, R.A. and Moffat, K. (2010) Structure and function [40] Takahashi, Y. and Ebrey, T.G. (2003) Molecular basis of spectral tuning in the of plant photoreceptors. Annu. Rev. Plant Biol.. newt short wavelength sensitive visual pigment. Biochemistry 42, 6025–6034. [15] Schleicher, E. et al. (2004) On the reaction mechanism of adduct formation in [41] Nash, A.I., Ko, W.H., Harper, S.M. and Gardner, K.H. (2008) A conserved LOV domains of the plant blue-light receptor phototropin. J. Am. Chem. Soc. glutamine plays a central role in LOV domain signal transmission and its 126, 11067–11076. duration. Biochemistry 47, 13842–13849. [16] Gauden, M., van Stokkum, I.H.M., Key, J.M., Luhrs, D.C., Van Grondelle, R., [42] Jones, M.A., Feeney, K.A., Kelly, S.M. and Christie, J.M. (2007) Mutational Hegemann, P. and Kennis, J.T.M. (2006) Hydrogen-bond switching through a analysis of phototropin 1 provides insights into the mechanism underlying radical pair mechanism in a flavin-binding photoreceptor. Proc. Nat. Acad. Sci. LOV2 signal transmission. J. Biol. Chem. 282, 6405–6414. USA 103, 10895–10900. [43] Hendrischk, A.K. et al. (2009) A cryptochrome-like protein is involved in the [17] Gauden, M., Yeremenko, S., Laan, W., van Stokkum, I.H.M., Ihalainen, J.A., van regulation of photosynthesis genes in Rhodobacter sphaeroides. Mol. Microbiol. Grondelle, R., Hellingwerf, K.J. and Kennis, J.T.M. (2005) Photocycle of the 74, 990–1003. flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of [44] Zirak, P., Penzkofer, A., Moldt, J., Pokorny, R., Batschauer, A. and Essen, L.O. photosynthesis genes. Biochemistry 44, 3653–3662. (2009) Photocycle dynamics of the E149A mutant of cryptochrome 3 from [18] Grinstead, J.S., Avila-Perez, M., Hellingwerf, K.J., Boelens, R. and Kaptein, R. Arabidopsis thaliana. J. Photochem. Photobiol. B-Biol. 97, 94–108. (2006) Light-induced flipping of a conserved glutamine sidechain and its [45] Zeugner, A., Byrdin, M., Bouly, J.P., Bakrim, N., Giovani, B., Brettel, K. and orientation in the AppA BLUF domain. J. Am. Chem. Soc. 128, 15066–15067. Ahmad, M. (2005) Light-induced electron transfer in Arabidopsis [19] Gauden, M. et al. (2007) On the role of aromatic side chains in the cryptochrome-1 correlates with in vivo function. J. Biol. Chem. 280, 19437– photoactivation of BLUF domains. Biochemistry 46, 7405–7415. 19440. 172 J.C. Arents et al. / FEBS Letters 585 (2011) 167–172

[46] Xia, W.Z., Wang, Q., Han, J., Zhao, C., Hong, Y., Zeng, L., Tang, L. and Ying, W. [49] Miller, G. (2006) Optogenetics: shining new light on neural circuits. Science (2009) Roles of NAD(+)/NADH and NADP(+)/NADPH in cell death. Curr. Pharm. 314, 1674–1676. Des. 15, 12–19. [50] Ernst, O.P., Murcia, P.A.S., Daldrop, P., Tsunoda, S.P., Kateriya, S. and [47] Lanzl, K., von Sanden-Flohe, M., Kutta, R.J. and Dick, B. (2010) Photoreaction of Hegemann, P. (2008) Photoactivation of channel rhodopsin. J. Biol. Chem. mutated LOV photoreceptor domains from Chlamydomonas reinhardtii with 283, 1637–1643. aliphatic mercaptans: implications for the mechanism of wild type LOV. Phys. [51] Moglich, A., Ayers, R.A. and Moffat, K. (2010) Addition at the molecular level: Chem. Chem. Phys. 12, 6594–6604. signal integration in designed Per-ARNT-Sim receptor proteins. J. Mol. Biol. [48] Kim, S.K., Mason, J.T., Knaff, D.B., Bauer, C.E. and Setterdahl, A.T. (2006) Redox 400, 477–486. properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA. Photosynth. Res. 89, 89–98.