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On the midpoint potential of the FAD chromophore in a BLUF-domain containing photoreceptor protein
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
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Article history: Received 16 May 2010 Revised 30 September 2010 Accepted 18 November 2010 Available online 24 November 2010 Edited by Richard Cogdell Keywords: LOV domain Cryptochrome Redox titration Site-directed mutation Reduced and oxidized FAD Flavin semiquinone
a b s t r a c t The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals 260 mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which 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. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The molecular basis by which living organisms respond to blue light has long remained a mystery, to the extent that the photosensory receptors involved in this response were generally referred to as ‘cryptic’ for several decades. Presumably this was in part due to the high-energy content of blue photons, which left little room – when varying light intensity – between detection of the biological response and infliction of irreversible damage. This situation has changed, first with the discovery of PYP and the xanthopsin family [1–3], and subsequently with the molecular (biological) characterization of three different families of flavin-containing photoreceptor families [4]. The first of these latter three families were the cryptochromes (Cry), of which a molecular (biological) characterization was provided by Cashmore and colleagues [5]. Subsequently, also LOV domains [6] and BLUF domains [7] were discovered and characterized [8,9]. This has led to a situation that by now of key representatives of each of these families the molecular structure and mechanism of primary photochemistry has been resolved and this knowledge has been the basis for further detailed subdivision of the Cry [10] and LOV families [11], as well as several interesting engineering applications (see e.g. [12,13]). ⇑ Corresponding author. Address: Laboratory for Microbiology, Swammerdam Institute for Life science, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Fax: +31 20 5257056. E-mail address: [email protected] (K.J. Hellingwerf).
These three flavin-containing photoreceptor families each use a characteristically different type of primary photochemistry (for a review see: [4,14]): Best characterized are the LOV domains, most of which bind FMN, rather than FAD, which is the physiological chromophore in the BLUF and Cry families. In the LOV domains light absorption leads to a singlet excited state, which converts through inter-system crossing to a triplet state on the nanosecond timescale. The triplet state then decays at the microsecond timescale and forms a state in which the C(4) atom of the isooxallazine ring of the flavin forms a covalent bond with the sulfur atom of a nearby cysteine side chain. The deformation of the flavin ring system that accompanies this covalent bond formation then leads to rearrangement of its surrounding amino acid side chains, amongst which is a key glutamine, that ‘flips’ because of this. It cannot yet be decided whether the covalent bond formation is preceded by hydrogen, electron and/or proton transfer [15]. In BLUF domains light absorption leads to reversible electron-, followed by proton- [16], or hydrogen- [17] transfer at the picosecond timescale, which then causes a similar ‘glutamine flip’ as in LOV domains, as was apparent not only from structural studies [18] but also suggested by trans-family engineering studies between 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 to the flipping glutamine, but other residues may function as a donor as well [19]. In Cryptochromes light absorption initiates 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
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oxidized FAD that is present in the dark. This presumably leads to a long-living bi-radical state that plays a key role in crypochrome signaling [20]. In all these three families the photochemically active species in the receptor state (i.e. the form present in the dark prior to light absorption) is the oxidized form of the FAD or FMN, as is apparent from the wavelength-dependence of their activation (see e.g. [21]), in spite of the fact that the intracellular environment in which such photoreceptors reside may become quite reduced, particularly when cells are confronted with a lack of oxygen. For two of the photoreceptor families the redox midpoint potential has recently been reported, i.e. for LOV domains [22] and Cryptochromes [23]. Here we report the same characteristic for the BLUF domain of AppA from Rhodobacter sphaeroides and several variants thereof, obtained through site-directed mutagenesis, and these results are compared with parallel measurements on selected LOV domains. The results obtained are discussed in the light of the mechanism of primary photochemistry in the three flavin-containing bluelight photoreceptor families and are compared with the redoxand photochemical characteristics of photolyase.
cuvette, argon was flushed over the reaction mixture at approximately 1 ml/min. To ensure redox equilibrium between the added dye and the flavoprotein during the measurements, the concentration of xanthine oxidase was kept low enough to allow the reduction of the components in the cuvette take at least two hours. Midpoint potentials (Em(protein)) can be calculated from such measurements e.g. when the oxidized- and reduced forms of the protein to be titrated have reached the same concentration, from the relation: ox
red
Em ðproteinÞ ¼ Em ðdyeÞ þ ð59=nÞ: logð½dye =½dye
Þ
ð1Þ
In this equation Em is the redox midpoint potential (expressed relative to the standard calomel electrode), and n the number of electrons involved in the redox transition. Either safranin O (Em = 289 mV), phenosafranin (Em = 252 mV) or anthraquinone-2-sulfonate (Em = 225 mV) was used as the indicator dye, with 2 lM methylviologen as a mediator. All these dyes are subject to a oneelectron redox transition. Time series of visible spectra were recorded using an HP8453 UV/visible spectrophotometer from 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 Xanthine oxidase (from buttermilk), dyes (anthraquinone-2sulfonate, methylviologen, phenosafranin and safranin O), nucleotides (FMN and FAD), buffers and general chemicals were bought from Sigma–Aldrich Co, St. Louis, MO, USA. Glucose oxidase (lyophilized) was supplied by Roche Products Ltd., Hertfordshireand, UK, and Argon gas (Argon 5.0 Instrument; >99.99% pure) was provided by Praxair (Vlaardingen, The Netherlands). 2.2. Purification of photoreceptor domains The BLUF domain of AppA from Rb. sphaeroides (AppA5-125) and several of its variants obtained through site-directed mutagenesis were isolated as previously described [24]. YtvA was purified using the procedure described in Avila Perez et al. [25]. The LOV2 domain of phototropin-1 from Avena sativa was isolated after heterologous over-expression in Escherichia coli as described elsewhere [26].
The W104A-, Q63N-, Y21F- and Q63H-variants of the BLUF domain of AppA were constructed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and pQEAppA5-125 as the template DNA for the PCR reactions [19]. All mutations were confirmed through DNA-sequence analysis. 2.5. Calculations Midpoint potentials were routinely calculated from the interception with the ordinate in a plot of the redox potential of the dye versus the redox potential of the protein under study. Occasionally, the time series of spectral data were de-convoluted with global analysis, prior to calculations of the concentration of the oxidized and reduced form of the photoreceptor protein. This did not significantly change the outcome of the calculations. Redox midpoint potentials of mediators and dyes were taken from [27]. 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 flavincontaining photoreceptor proteins was adapted from the xanthine/ xanthine oxidase method that was developed by Massey [27,28]. UV/visible spectra were recorded in a 3 ml quartz cuvette which could be closed with a screw cap and containing a septum to prevent oxygen inflow and a small stirrer bean to achieve mixing of its contents within a few seconds. The reaction mixture was kept at room temperature with a forced air-flow through a cooling mantel that surrounded the cuvette. All solutions were pre-treated by bubbling with argon for at least an hour prior to spectroscopic measurements. The standard assay buffer contained 50 mM Tris– HCl of pH = 8.0, 1 mM EDTA, 400 lM xanthine, 2 lM methylviologen, 20 lM of the particular dye, 10 mM glucose and 20 lM of the particular flavoprotein in a total volume of 2 ml. After addition 10 ll of glucose oxidase (from a stock solution on 10 mg/ml), the reversible reduction of the flavoprotein was initiated with 10 ll of xanthine oxidase (freshly prepared from a stock solution of 15 mg/ml). Glucose and glucose oxidase were then added to remove molecular oxygen and keep the reaction mixture anaerobic. Simultaneously, via needles punched through the septum of the
BLUF domains, as isolated and purified from their natural host, as well as after heterologous (over)expression in E. coli, have their flavin in the fully oxidized form [7,30]. We therefore expect the BLUF domains to gradually be converted to the reduced form under our measurement conditions. As the indicator/reporter probe several dyes were tested, using the selection criteria of: (i) minimal spectral overlap between the dye and the oxidized and reduced form of the photoreceptor protein, and (ii) close match of the redox midpoint potential of the selected dye and the particular flavoprotein under study. Using these criteria, particularly phenosafranin, safranin O and anthraquinone-2-sulfonate (all three used at 20 lM) were found to be suitable. In addition, in all experiments 2 lM methylviologen was used as a mediator between the xanthine oxidase, the flavin-containing photosensory receptor and the indicator dye. The initial experiments that we carried out showed poor reproducibility. This has most likely been caused by slow diffusion of molecular oxygen into the cuvette and could neither be prevented by tighter fitting of its cap, nor by more intense flushing with argon. We therefore decided to add the combination of glucose
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plus glucose oxidase to the contents of the cuvette. The high affinity and turnover capacity of the latter enzyme for molecular oxygen (200 lM and 250 U/mg; see product sheet of the enzyme) decreases the remaining oxygen level to very low values and may have prevented the formation of reactive oxygen species by xanthine oxidase [31]. In this configuration a number of indicator dyes were tested, like anthraquinone-2-sulfonate, phenosafranine, and safranine O. Of these dyes, particularly phenosafranine is excellently suited for the titration of most of the BLUF domains. For the LOV domains, which have a more negative redox-midpoint potential (see further below), both safranine O and benzyl viologen can be used. By limiting the amount of xantine oxidase to 75 lg/ml (final concentration) sufficiently slow reduction is achieved so that it will take more than 2 h before the majority of the flavoprotein has been reduced (Fig. 1A and B). By recording absorbance changes at 521 and 446 nm it is then possible (after correction for the contribution of the absorbance changes at 446 nm by the phenosafranine dye) to
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calculate the oxidized/reduced ratio of both components. Plotting these ratio’s against each other then allows one to calculate the redox-midpoint potential of the flavoprotein photoreceptor from the known midpoint potential of the indicator dye (252 mV; see Section 2.3). These calculations are most conveniently carried out by using the values of the intersection of the titration curve with either abscise or ordinate (Fig. 1C). Registration of complete spectra (e.g. from 300 to 650 nm) at regular intervals, followed by global analysis of such data (see e.g. [29]), also allows one to calculate the concentration of the oxidized and reduced forms of the dye and the flavoprotein under study. This approach, however, did not lead to significantly different results (data not shown). To further test the redox equilibration between the various redox couples in the sample cuvette during a titration, in a subsequent experiment, after reduction of the sample with xantine and xantine oxidase, the cap of the cuvette was removed. To test the reversibility of sample reduction with xanthine 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 flavincontaining 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 recordings 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 280 to 700 nm) of the color changes in the reaction mixture during the reducing titration. Spectra were taken at 30 s intervals, but only every tenth spectrum is shown. (C) Plot of the redox potential of the BLUF domain versus that of the dye, from which the redox midpoint potential can be calculated according to the method described in the Secton 2.
Fig. 2. Reversibility of the reduction and oxidation of the BLUF domain of AppA, with xanthine plus xanthine oxidase and with oxygen, respectively. Data were obtained with 20 lM of the wild type AppA5-125 domain. The reduction reaction was recorded in a period of 5000 s; the oxidation reaction was initiated by opening the lid of the cuvette and took 500 s. Solid line: oxidation; dashed line: reduction of the flavoprotein. ‘dye’: phenosafranin.
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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 photoreceptors YtvA and Phototropin 1. For details: see Section 3 and 4. Bars represent standard deviations. N = number of independent assays of the particular midpoint potential. Grey: BLUF domains; black: LOV domains; open bar: aqueous solution of 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 AppA from Rhodobacter sphaeroides.
(e.g. glutamine-63) and in the Y21F/W104F double mutant [19], the midpoint potential of the protein-bound flavin shifts to a value close to the midpoint potential for free flavin in aqueous solution. In none of these redox titrations evidence for the formation of a flavosemiquinone intermediate form has been obtained. 4. Discussion The xanthine/xanthine oxidase method [27,28] that we selected for the redox titrations reported in this investigation worked well, as can be concluded from the absence of hysteresis in Fig. 2. Besides the general problem of slow equilibration, which is inherent to many redox reactions, another crucial factor in these experiments is the exclusion of molecular oxygen. Particularly in the most negative range of redox potentials studied, flushing with argon did not suffice to achieve this. Addition of glucose/glucose oxidase, however, was sufficient to solve this problem. Besides BLUF and LOV domains (see Section 3.2), values for the redox-midpoint potential of Cryptochromes and photolyases are 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 cryptochromes (see also Section 1). The very low midpoint potentials of LOV domains may make them very robust and on this basis may explain their very wide phylogenetic distribution, even in very extremophilic microorganisms [11].
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 specific 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 observation is the rule and which is the exception. In the receptor state of the photosensory receptor proteins the latter of the three factors seems to make the most important contribution. A change in the number of hydrogen bonds to the aromatic ring of the flavin is expected to shift its UV/Vis [39–42]. Accordingly, one would also expect a correlation between the redox 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 Fig. 4). Although several cryptochromes have their absorbance maximum at even shorter wavelength than 440 nm [43,44]), also longer wavelength maxima have been reported for this photoreceptor domain (e.g. [45]), and also free FAD does not show this correlation. Significantly, flavin both in aqueous solution and in the Cry domain, has largely lost its vibrational fine structure, which may explain this lack of correlation. An intriguing open question that remains is the relation between the intracellular redox potential and the in vivo redox state of these flavin-containing photoreceptors. The intracellular redox potential differs a lot between the various relevant redox couples (e.g. between NAD+/NADH and NADP+/NADPH, but for this discussion the former couple presumably is the most important, because of the abundance of this nucleotide. In the eukaryotic cytoplasm the redox potential of this couple most likely is higher than 300 mV ([46]. Based on this, one can easily rationalize that LOV domains in vivo will have an oxidized flavin in the receptor state. However, for BLUF-, and particularly for CRY domains this is less clear. Modulation of the redox state of their flavin chromophore in the receptor state may therefore form part of the regulation to
Fig. 4. Correlation between absorbance maximum and redox midpoint potential 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 purified proteins. Symbols represent: : YtvA; }: AsLOV2; N: AppA5-125Q63 N; 4: AppA5-125Y21F; j: AppA5-125W104A; h: AppA5-125; d: AppA5-125Q63H; s: AppA5125Y21F W104F.
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which their activity is subjected (compare [47]). In this respect it is relevant to note that the midpoint potential of the sulfhydryl groups of AppA, which mediate the redox regulation of its interaction with PpsR, has been estimated to be 315 mV [48]. The cytoplasmic redox potential may particularly have an effect in prokaryotes, as many of them can thrive in the absence of oxygen, i.e. in very reducing conditions. Therefore, a lot remains to be learned about the mechanism of integration of light- and redox signals, particularly in BLUF domains. Of particular relevance is the question whether or not light-sensitivity of flavin photoreceptors is strictly limited to receptor states with an oxidized flavin. Recently, the term ‘optogenetics’ was coined to refer to studies in which engineered photoreceptors are used to study a wide range of biological problems [49,50]. We anticipate that this field will bring more light to the details of this integration [51]. Knowledge to be gained in such studies may further aid engineering efforts, like the transformation of BLUF domains into LOV domains and visa versa [12], because they form the ultimate proof of proper understanding these photosensory receptor systems. References [1] Meyer, T.E. (1985) Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila. Biochim. Biophys. Acta 806, 175–183. [2] Sprenger, W.W., Hoff, W.D., Armitage, J.P. and Hellingwerf, K.J. (1993) The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J. Bacteriol. 175, 3096–3104. [3] Kort, R. et al. (1996) The xanthopsins: a new family of eubacterial blue-light photoreceptors. EMBO J. 15, 3209–3218. [4] van der Horst, M.A. and Hellingwerf, K.J. (2004) Photoreceptor proteins, ‘‘star actors of modern times’’: a review of the functional dynamics in the structure of representative members of six different photoreceptor families. Acc. Chem. Res. 37, 13–20. [5] Ahmad, M. and Cashmore, A.R. (1993) HY4 gene of A. Thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, 162– 166. [6] Huala, E., Oeller, P.W., Liscum, E., Han, I.S., Larsen, E. and Briggs, W.R. (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278, 2120–2123. [7] Masuda, S. and Bauer, C.E. (2002) AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110, 613–623. [8] Christie, J.M., Salomon, M., Nozue, K., Wada, M. and Briggs, W.R. (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc. Natl. Acad. Sci. USA 96, 8779–8783. [9] Gomelsky, M. and Klug, G. (2002) BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms. Trends Biochem. Sci. 27, 497–500. [10] Lin, C. and Todo, T. (2005) The cryptochromes. Genome Biol. 6, 220. [11] Krauss, U., Minh, B.Q., Losi, A., Gartner, W., Eggert, T., von Haeseler, A. and Jaeger, K.E. (2009) Distribution and phylogeny of light-oxygen-voltage-bluelight-signaling proteins in the three kingdoms of life. J. Bacteriol. 191, 7234– 7242. [12] Suzuki, H., Okajima, K., Ikeuchi, M. and Noguchi, T. (2008) LOV-like flavin-Cys adduct formation by introducing a Cys residue in the BLUF domain of TePixD. J. Am. Chem. Soc. 130, 12884. [13] Moglich, A., Ayers, R.A. and Moffat, K. (2009) Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol. 385, 1433–1444. [14] Moglich, A., Yang, X., Ayers, R.A. and Moffat, K. (2010) Structure and function of plant photoreceptors. Annu. Rev. Plant Biol.. [15] Schleicher, E. et al. (2004) On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin. J. Am. Chem. Soc. 126, 11067–11076. [16] Gauden, M., van Stokkum, I.H.M., Key, J.M., Luhrs, D.C., Van Grondelle, R., Hegemann, P. and Kennis, J.T.M. (2006) Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor. Proc. Nat. Acad. Sci. USA 103, 10895–10900. [17] Gauden, M., Yeremenko, S., Laan, W., van Stokkum, I.H.M., Ihalainen, J.A., van Grondelle, R., Hellingwerf, K.J. and Kennis, J.T.M. (2005) Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes. Biochemistry 44, 3653–3662. [18] Grinstead, J.S., Avila-Perez, M., Hellingwerf, K.J., Boelens, R. and Kaptein, R. (2006) Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain. J. Am. Chem. Soc. 128, 15066–15067. [19] Gauden, M. et al. (2007) On the role of aromatic side chains in the photoactivation of BLUF domains. Biochemistry 46, 7405–7415.
171
[20] Bouly, J.P. et al. (2007) Cryptochrome blue light photoreceptors are activated 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 sigma(B)-dependent stress response of Bacillus subtilis via YtvA. J. Bacteriol. 188, 6411–6414. [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 photochemical reduction, and influence on the photocycle. ChemBioChem 8, 2256–2264. [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 spectroelectrochemical comparison of the flavin redox transitions. J. Am. Chem. Soc. 131, 426–427. [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-lightusing flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide 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 Hellingwerf, K. (2009) In vivo mutational analysis of YtvA from Bacillus subtilis mechanism of light activation of the general stress response. J. Biol. Chem. 284, 24958–24964. [26] Harper, S.M., Neil, L.C. and Gardner, K.H. (2003) Structural basis of a 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– 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) Covalent flavinylation is essential for efficient redox catalysis in vanillylalcohol oxidase. J. Biol. Chem. 274, 35514–35520. [29] Hendriks, J. and Hellingwerf, K.J. (2009) PH dependence of the photoactive yellow protein photocycle recovery reaction reveals a new late photocycle intermediate with a deprotonated chromophore. J. Biol. Chem. 284, 5277– 5288. [30] Laan, W., Bednarz, T., Heberle, J. and Hellingwerf, K.J. (2004) Chromophore composition of a heterologously expressed BLUF-domain. Photochem. Photobiol. Sci. 3, 1011–1016. [31] Zanotto-Filho, A., Schroder, R. and Moreira, J.C. (2008) Xanthine oxidasedependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells. Free Radic. Res. 42, 593–601. [32] Segel, H. (1986) Biochemical Calculations, 2nd ed, John Wiley and Sons Inc., New York. [33] Berg, J.M., Tymoczko, J.L. and Stryer, L. (2007) Biochemistry (sixth edition), W.H. Freeman and Company, New York. [34] Lin, C.T., Robertson, D.E., Ahmad, M., Raibekas, A.A., Jorns, M.S., Dutton, P.L. and Cashmore, A.R. (1995) Association of flavin adenine-dinucleotide with the Arabidopsis blue-light receptor Cry1. Science 269, 968–970. [35] Sokolowsky, K. et al. (2010) Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae Cryptochrome 1. J. Phys. Chem. B 114, 7121–7130. [36] Zhou, Z.M. and Swenson, R.P. (1996) The cumulative electrostatic effect of aromatic stacking interactions and the negative electrostatic environment of the flavin mononucleotide binding site is a major determinant of the reduction potential for the flavodoxin from Desulfovibrio vulgaris [Hildenborough]. Biochemistry 35, 15980–15988. [37] Bhattacharyya, S., Stankovich, M.T., Truhlar, D.G. and Gao, J.L. (2007) Combined quantum mechanical and molecular mechanical simulations of one- and two-electron reduction potentials of flavin cofactor in water, medium-chain acyl-CoA dehydrogenase, and cholesterol oxidase. J. Phys. Chem. A 111, 5729–5742. [38] Chang, F.C., Bradley, L.H. and Swenson, R.P. (2001) Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin. Biochimica Et Biophysica Acta-Bioenergetics 1504, 319–328. [39] Nozaki, D., Iwata, T., Ishikawa, T., Todo, T., Tokutomi, S. and Kandori, H. (2004) Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3. Biochemistry 43, 8373–8379. [40] Takahashi, Y. and Ebrey, T.G. (2003) Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment. Biochemistry 42, 6025–6034. [41] Nash, A.I., Ko, W.H., Harper, S.M. and Gardner, K.H. (2008) A conserved glutamine plays a central role in LOV domain signal transmission and its duration. Biochemistry 47, 13842–13849. [42] Jones, M.A., Feeney, K.A., Kelly, S.M. and Christie, J.M. (2007) Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission. J. Biol. Chem. 282, 6405–6414. [43] Hendrischk, A.K. et al. (2009) A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides. Mol. Microbiol. 74, 990–1003. [44] Zirak, P., Penzkofer, A., Moldt, J., Pokorny, R., Batschauer, A. and Essen, L.O. (2009) Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana. J. Photochem. Photobiol. B-Biol. 97, 94–108. [45] Zeugner, A., Byrdin, M., Bouly, J.P., Bakrim, N., Giovani, B., Brettel, K. and Ahmad, M. (2005) Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function. J. Biol. Chem. 280, 19437– 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. (2009) Roles of NAD(+)/NADH and NADP(+)/NADPH in cell death. Curr. Pharm. Des. 15, 12–19. [47] Lanzl, K., von Sanden-Flohe, M., Kutta, R.J. and Dick, B. (2010) Photoreaction of mutated LOV photoreceptor domains from Chlamydomonas reinhardtii with aliphatic mercaptans: implications for the mechanism of wild type LOV. Phys. Chem. Chem. Phys. 12, 6594–6604. [48] Kim, S.K., Mason, J.T., Knaff, D.B., Bauer, C.E. and Setterdahl, A.T. (2006) Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA. Photosynth. Res. 89, 89–98.
[49] Miller, G. (2006) Optogenetics: shining new light on neural circuits. Science 314, 1674–1676. [50] Ernst, O.P., Murcia, P.A.S., Daldrop, P., Tsunoda, S.P., Kateriya, S. and Hegemann, P. (2008) Photoactivation of channel rhodopsin. J. Biol. Chem. 283, 1637–1643. [51] Moglich, A., Ayers, R.A. and Moffat, K. (2010) Addition at the molecular level: signal integration in designed Per-ARNT-Sim receptor proteins. J. Mol. Biol. 400, 477–486.
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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
a r t i c l e
i n f o
Article history: Received 16 May 2010 Revised 30 September 2010 Accepted 18 November 2010 Available online 24 November 2010 Edited by Richard Cogdell Keywords: LOV domain Cryptochrome Redox titration Site-directed mutation Reduced and oxidized FAD Flavin semiquinone
a b s t r a c t The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals 260 mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which 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. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The molecular basis by which living organisms respond to blue light has long remained a mystery, to the extent that the photosensory receptors involved in this response were generally referred to as ‘cryptic’ for several decades. Presumably this was in part due to the high-energy content of blue photons, which left little room – when varying light intensity – between detection of the biological response and infliction of irreversible damage. This situation has changed, first with the discovery of PYP and the xanthopsin family [1–3], and subsequently with the molecular (biological) characterization of three different families of flavin-containing photoreceptor families [4]. The first of these latter three families were the cryptochromes (Cry), of which a molecular (biological) characterization was provided by Cashmore and colleagues [5]. Subsequently, also LOV domains [6] and BLUF domains [7] were discovered and characterized [8,9]. This has led to a situation that by now of key representatives of each of these families the molecular structure and mechanism of primary photochemistry has been resolved and this knowledge has been the basis for further detailed subdivision of the Cry [10] and LOV families [11], as well as several interesting engineering applications (see e.g. [12,13]). ⇑ Corresponding author. Address: Laboratory for Microbiology, Swammerdam Institute for Life science, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Fax: +31 20 5257056. E-mail address: [email protected] (K.J. Hellingwerf).
These three flavin-containing photoreceptor families each use a characteristically different type of primary photochemistry (for a review see: [4,14]): Best characterized are the LOV domains, most of which bind FMN, rather than FAD, which is the physiological chromophore in the BLUF and Cry families. In the LOV domains light absorption leads to a singlet excited state, which converts through inter-system crossing to a triplet state on the nanosecond timescale. The triplet state then decays at the microsecond timescale and forms a state in which the C(4) atom of the isooxallazine ring of the flavin forms a covalent bond with the sulfur atom of a nearby cysteine side chain. The deformation of the flavin ring system that accompanies this covalent bond formation then leads to rearrangement of its surrounding amino acid side chains, amongst which is a key glutamine, that ‘flips’ because of this. It cannot yet be decided whether the covalent bond formation is preceded by hydrogen, electron and/or proton transfer [15]. In BLUF domains light absorption leads to reversible electron-, followed by proton- [16], or hydrogen- [17] transfer at the picosecond timescale, which then causes a similar ‘glutamine flip’ as in LOV domains, as was apparent not only from structural studies [18] but also suggested by trans-family engineering studies between 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 to the flipping glutamine, but other residues may function as a donor as well [19]. In Cryptochromes light absorption initiates 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
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oxidized FAD that is present in the dark. This presumably leads to a long-living bi-radical state that plays a key role in crypochrome signaling [20]. In all these three families the photochemically active species in the receptor state (i.e. the form present in the dark prior to light absorption) is the oxidized form of the FAD or FMN, as is apparent from the wavelength-dependence of their activation (see e.g. [21]), in spite of the fact that the intracellular environment in which such photoreceptors reside may become quite reduced, particularly when cells are confronted with a lack of oxygen. For two of the photoreceptor families the redox midpoint potential has recently been reported, i.e. for LOV domains [22] and Cryptochromes [23]. Here we report the same characteristic for the BLUF domain of AppA from Rhodobacter sphaeroides and several variants thereof, obtained through site-directed mutagenesis, and these results are compared with parallel measurements on selected LOV domains. The results obtained are discussed in the light of the mechanism of primary photochemistry in the three flavin-containing bluelight photoreceptor families and are compared with the redoxand photochemical characteristics of photolyase.
cuvette, argon was flushed over the reaction mixture at approximately 1 ml/min. To ensure redox equilibrium between the added dye and the flavoprotein during the measurements, the concentration of xanthine oxidase was kept low enough to allow the reduction of the components in the cuvette take at least two hours. Midpoint potentials (Em(protein)) can be calculated from such measurements e.g. when the oxidized- and reduced forms of the protein to be titrated have reached the same concentration, from the relation: ox
red
Em ðproteinÞ ¼ Em ðdyeÞ þ ð59=nÞ: logð½dye =½dye
Þ
ð1Þ
In this equation Em is the redox midpoint potential (expressed relative to the standard calomel electrode), and n the number of electrons involved in the redox transition. Either safranin O (Em = 289 mV), phenosafranin (Em = 252 mV) or anthraquinone-2-sulfonate (Em = 225 mV) was used as the indicator dye, with 2 lM methylviologen as a mediator. All these dyes are subject to a oneelectron redox transition. Time series of visible spectra were recorded using an HP8453 UV/visible spectrophotometer from 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 Xanthine oxidase (from buttermilk), dyes (anthraquinone-2sulfonate, methylviologen, phenosafranin and safranin O), nucleotides (FMN and FAD), buffers and general chemicals were bought from Sigma–Aldrich Co, St. Louis, MO, USA. Glucose oxidase (lyophilized) was supplied by Roche Products Ltd., Hertfordshireand, UK, and Argon gas (Argon 5.0 Instrument; >99.99% pure) was provided by Praxair (Vlaardingen, The Netherlands). 2.2. Purification of photoreceptor domains The BLUF domain of AppA from Rb. sphaeroides (AppA5-125) and several of its variants obtained through site-directed mutagenesis were isolated as previously described [24]. YtvA was purified using the procedure described in Avila Perez et al. [25]. The LOV2 domain of phototropin-1 from Avena sativa was isolated after heterologous over-expression in Escherichia coli as described elsewhere [26].
The W104A-, Q63N-, Y21F- and Q63H-variants of the BLUF domain of AppA were constructed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and pQEAppA5-125 as the template DNA for the PCR reactions [19]. All mutations were confirmed through DNA-sequence analysis. 2.5. Calculations Midpoint potentials were routinely calculated from the interception with the ordinate in a plot of the redox potential of the dye versus the redox potential of the protein under study. Occasionally, the time series of spectral data were de-convoluted with global analysis, prior to calculations of the concentration of the oxidized and reduced form of the photoreceptor protein. This did not significantly change the outcome of the calculations. Redox midpoint potentials of mediators and dyes were taken from [27]. 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 flavincontaining photoreceptor proteins was adapted from the xanthine/ xanthine oxidase method that was developed by Massey [27,28]. UV/visible spectra were recorded in a 3 ml quartz cuvette which could be closed with a screw cap and containing a septum to prevent oxygen inflow and a small stirrer bean to achieve mixing of its contents within a few seconds. The reaction mixture was kept at room temperature with a forced air-flow through a cooling mantel that surrounded the cuvette. All solutions were pre-treated by bubbling with argon for at least an hour prior to spectroscopic measurements. The standard assay buffer contained 50 mM Tris– HCl of pH = 8.0, 1 mM EDTA, 400 lM xanthine, 2 lM methylviologen, 20 lM of the particular dye, 10 mM glucose and 20 lM of the particular flavoprotein in a total volume of 2 ml. After addition 10 ll of glucose oxidase (from a stock solution on 10 mg/ml), the reversible reduction of the flavoprotein was initiated with 10 ll of xanthine oxidase (freshly prepared from a stock solution of 15 mg/ml). Glucose and glucose oxidase were then added to remove molecular oxygen and keep the reaction mixture anaerobic. Simultaneously, via needles punched through the septum of the
BLUF domains, as isolated and purified from their natural host, as well as after heterologous (over)expression in E. coli, have their flavin in the fully oxidized form [7,30]. We therefore expect the BLUF domains to gradually be converted to the reduced form under our measurement conditions. As the indicator/reporter probe several dyes were tested, using the selection criteria of: (i) minimal spectral overlap between the dye and the oxidized and reduced form of the photoreceptor protein, and (ii) close match of the redox midpoint potential of the selected dye and the particular flavoprotein under study. Using these criteria, particularly phenosafranin, safranin O and anthraquinone-2-sulfonate (all three used at 20 lM) were found to be suitable. In addition, in all experiments 2 lM methylviologen was used as a mediator between the xanthine oxidase, the flavin-containing photosensory receptor and the indicator dye. The initial experiments that we carried out showed poor reproducibility. This has most likely been caused by slow diffusion of molecular oxygen into the cuvette and could neither be prevented by tighter fitting of its cap, nor by more intense flushing with argon. We therefore decided to add the combination of glucose
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plus glucose oxidase to the contents of the cuvette. The high affinity and turnover capacity of the latter enzyme for molecular oxygen (200 lM and 250 U/mg; see product sheet of the enzyme) decreases the remaining oxygen level to very low values and may have prevented the formation of reactive oxygen species by xanthine oxidase [31]. In this configuration a number of indicator dyes were tested, like anthraquinone-2-sulfonate, phenosafranine, and safranine O. Of these dyes, particularly phenosafranine is excellently suited for the titration of most of the BLUF domains. For the LOV domains, which have a more negative redox-midpoint potential (see further below), both safranine O and benzyl viologen can be used. By limiting the amount of xantine oxidase to 75 lg/ml (final concentration) sufficiently slow reduction is achieved so that it will take more than 2 h before the majority of the flavoprotein has been reduced (Fig. 1A and B). By recording absorbance changes at 521 and 446 nm it is then possible (after correction for the contribution of the absorbance changes at 446 nm by the phenosafranine dye) to
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calculate the oxidized/reduced ratio of both components. Plotting these ratio’s against each other then allows one to calculate the redox-midpoint potential of the flavoprotein photoreceptor from the known midpoint potential of the indicator dye (252 mV; see Section 2.3). These calculations are most conveniently carried out by using the values of the intersection of the titration curve with either abscise or ordinate (Fig. 1C). Registration of complete spectra (e.g. from 300 to 650 nm) at regular intervals, followed by global analysis of such data (see e.g. [29]), also allows one to calculate the concentration of the oxidized and reduced forms of the dye and the flavoprotein under study. This approach, however, did not lead to significantly different results (data not shown). To further test the redox equilibration between the various redox couples in the sample cuvette during a titration, in a subsequent experiment, after reduction of the sample with xantine and xantine oxidase, the cap of the cuvette was removed. To test the reversibility of sample reduction with xanthine 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 flavincontaining 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 recordings 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 280 to 700 nm) of the color changes in the reaction mixture during the reducing titration. Spectra were taken at 30 s intervals, but only every tenth spectrum is shown. (C) Plot of the redox potential of the BLUF domain versus that of the dye, from which the redox midpoint potential can be calculated according to the method described in the Secton 2.
Fig. 2. Reversibility of the reduction and oxidation of the BLUF domain of AppA, with xanthine plus xanthine oxidase and with oxygen, respectively. Data were obtained with 20 lM of the wild type AppA5-125 domain. The reduction reaction was recorded in a period of 5000 s; the oxidation reaction was initiated by opening the lid of the cuvette and took 500 s. Solid line: oxidation; dashed line: reduction of the flavoprotein. ‘dye’: phenosafranin.
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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 photoreceptors YtvA and Phototropin 1. For details: see Section 3 and 4. Bars represent standard deviations. N = number of independent assays of the particular midpoint potential. Grey: BLUF domains; black: LOV domains; open bar: aqueous solution of 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 AppA from Rhodobacter sphaeroides.
(e.g. glutamine-63) and in the Y21F/W104F double mutant [19], the midpoint potential of the protein-bound flavin shifts to a value close to the midpoint potential for free flavin in aqueous solution. In none of these redox titrations evidence for the formation of a flavosemiquinone intermediate form has been obtained. 4. Discussion The xanthine/xanthine oxidase method [27,28] that we selected for the redox titrations reported in this investigation worked well, as can be concluded from the absence of hysteresis in Fig. 2. Besides the general problem of slow equilibration, which is inherent to many redox reactions, another crucial factor in these experiments is the exclusion of molecular oxygen. Particularly in the most negative range of redox potentials studied, flushing with argon did not suffice to achieve this. Addition of glucose/glucose oxidase, however, was sufficient to solve this problem. Besides BLUF and LOV domains (see Section 3.2), values for the redox-midpoint potential of Cryptochromes and photolyases are 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 cryptochromes (see also Section 1). The very low midpoint potentials of LOV domains may make them very robust and on this basis may explain their very wide phylogenetic distribution, even in very extremophilic microorganisms [11].
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 specific 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 observation is the rule and which is the exception. In the receptor state of the photosensory receptor proteins the latter of the three factors seems to make the most important contribution. A change in the number of hydrogen bonds to the aromatic ring of the flavin is expected to shift its UV/Vis [39–42]. Accordingly, one would also expect a correlation between the redox 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 Fig. 4). Although several cryptochromes have their absorbance maximum at even shorter wavelength than 440 nm [43,44]), also longer wavelength maxima have been reported for this photoreceptor domain (e.g. [45]), and also free FAD does not show this correlation. Significantly, flavin both in aqueous solution and in the Cry domain, has largely lost its vibrational fine structure, which may explain this lack of correlation. An intriguing open question that remains is the relation between the intracellular redox potential and the in vivo redox state of these flavin-containing photoreceptors. The intracellular redox potential differs a lot between the various relevant redox couples (e.g. between NAD+/NADH and NADP+/NADPH, but for this discussion the former couple presumably is the most important, because of the abundance of this nucleotide. In the eukaryotic cytoplasm the redox potential of this couple most likely is higher than 300 mV ([46]. Based on this, one can easily rationalize that LOV domains in vivo will have an oxidized flavin in the receptor state. However, for BLUF-, and particularly for CRY domains this is less clear. Modulation of the redox state of their flavin chromophore in the receptor state may therefore form part of the regulation to
Fig. 4. Correlation between absorbance maximum and redox midpoint potential 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 purified proteins. Symbols represent: : YtvA; }: AsLOV2; N: AppA5-125Q63 N; 4: AppA5-125Y21F; j: AppA5-125W104A; h: AppA5-125; d: AppA5-125Q63H; s: AppA5125Y21F W104F.
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which their activity is subjected (compare [47]). In this respect it is relevant to note that the midpoint potential of the sulfhydryl groups of AppA, which mediate the redox regulation of its interaction with PpsR, has been estimated to be 315 mV [48]. The cytoplasmic redox potential may particularly have an effect in prokaryotes, as many of them can thrive in the absence of oxygen, i.e. in very reducing conditions. Therefore, a lot remains to be learned about the mechanism of integration of light- and redox signals, particularly in BLUF domains. Of particular relevance is the question whether or not light-sensitivity of flavin photoreceptors is strictly limited to receptor states with an oxidized flavin. Recently, the term ‘optogenetics’ was coined to refer to studies in which engineered photoreceptors are used to study a wide range of biological problems [49,50]. We anticipate that this field will bring more light to the details of this integration [51]. Knowledge to be gained in such studies may further aid engineering efforts, like the transformation of BLUF domains into LOV domains and visa versa [12], because they form the ultimate proof of proper understanding these photosensory receptor systems. References [1] Meyer, T.E. (1985) Isolation and characterization of soluble cytochromes, ferredoxins and other chromophoric proteins from the halophilic phototrophic bacterium Ectothiorhodospira halophila. Biochim. Biophys. Acta 806, 175–183. [2] Sprenger, W.W., Hoff, W.D., Armitage, J.P. and Hellingwerf, K.J. (1993) The eubacterium Ectothiorhodospira halophila is negatively phototactic, with a wavelength dependence that fits the absorption spectrum of the photoactive yellow protein. J. Bacteriol. 175, 3096–3104. [3] Kort, R. et al. (1996) The xanthopsins: a new family of eubacterial blue-light photoreceptors. EMBO J. 15, 3209–3218. [4] van der Horst, M.A. and Hellingwerf, K.J. (2004) Photoreceptor proteins, ‘‘star actors of modern times’’: a review of the functional dynamics in the structure of representative members of six different photoreceptor families. Acc. Chem. Res. 37, 13–20. [5] Ahmad, M. and Cashmore, A.R. (1993) HY4 gene of A. Thaliana encodes a protein with characteristics of a blue-light photoreceptor. Nature 366, 162– 166. [6] Huala, E., Oeller, P.W., Liscum, E., Han, I.S., Larsen, E. and Briggs, W.R. (1997) Arabidopsis NPH1: a protein kinase with a putative redox-sensing domain. Science 278, 2120–2123. [7] Masuda, S. and Bauer, C.E. (2002) AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides. Cell 110, 613–623. [8] Christie, J.M., Salomon, M., Nozue, K., Wada, M. and Briggs, W.R. (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc. Natl. Acad. Sci. USA 96, 8779–8783. [9] Gomelsky, M. and Klug, G. (2002) BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms. Trends Biochem. Sci. 27, 497–500. [10] Lin, C. and Todo, T. (2005) The cryptochromes. Genome Biol. 6, 220. [11] Krauss, U., Minh, B.Q., Losi, A., Gartner, W., Eggert, T., von Haeseler, A. and Jaeger, K.E. (2009) Distribution and phylogeny of light-oxygen-voltage-bluelight-signaling proteins in the three kingdoms of life. J. Bacteriol. 191, 7234– 7242. [12] Suzuki, H., Okajima, K., Ikeuchi, M. and Noguchi, T. (2008) LOV-like flavin-Cys adduct formation by introducing a Cys residue in the BLUF domain of TePixD. J. Am. Chem. Soc. 130, 12884. [13] Moglich, A., Ayers, R.A. and Moffat, K. (2009) Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol. 385, 1433–1444. [14] Moglich, A., Yang, X., Ayers, R.A. and Moffat, K. (2010) Structure and function of plant photoreceptors. Annu. Rev. Plant Biol.. [15] Schleicher, E. et al. (2004) On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin. J. Am. Chem. Soc. 126, 11067–11076. [16] Gauden, M., van Stokkum, I.H.M., Key, J.M., Luhrs, D.C., Van Grondelle, R., Hegemann, P. and Kennis, J.T.M. (2006) Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor. Proc. Nat. Acad. Sci. USA 103, 10895–10900. [17] Gauden, M., Yeremenko, S., Laan, W., van Stokkum, I.H.M., Ihalainen, J.A., van Grondelle, R., Hellingwerf, K.J. and Kennis, J.T.M. (2005) Photocycle of the flavin-binding photoreceptor AppA, a bacterial transcriptional antirepressor of photosynthesis genes. Biochemistry 44, 3653–3662. [18] Grinstead, J.S., Avila-Perez, M., Hellingwerf, K.J., Boelens, R. and Kaptein, R. (2006) Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain. J. Am. Chem. Soc. 128, 15066–15067. [19] Gauden, M. et al. (2007) On the role of aromatic side chains in the photoactivation of BLUF domains. Biochemistry 46, 7405–7415.
171
[20] Bouly, J.P. et al. (2007) Cryptochrome blue light photoreceptors are activated 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 sigma(B)-dependent stress response of Bacillus subtilis via YtvA. J. Bacteriol. 188, 6411–6414. [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 photochemical reduction, and influence on the photocycle. ChemBioChem 8, 2256–2264. [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 spectroelectrochemical comparison of the flavin redox transitions. J. Am. Chem. Soc. 131, 426–427. [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-lightusing flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide 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 Hellingwerf, K. (2009) In vivo mutational analysis of YtvA from Bacillus subtilis mechanism of light activation of the general stress response. J. Biol. Chem. 284, 24958–24964. [26] Harper, S.M., Neil, L.C. and Gardner, K.H. (2003) Structural basis of a 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– 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) Covalent flavinylation is essential for efficient redox catalysis in vanillylalcohol oxidase. J. Biol. Chem. 274, 35514–35520. [29] Hendriks, J. and Hellingwerf, K.J. (2009) PH dependence of the photoactive yellow protein photocycle recovery reaction reveals a new late photocycle intermediate with a deprotonated chromophore. J. Biol. Chem. 284, 5277– 5288. [30] Laan, W., Bednarz, T., Heberle, J. and Hellingwerf, K.J. (2004) Chromophore composition of a heterologously expressed BLUF-domain. Photochem. Photobiol. Sci. 3, 1011–1016. [31] Zanotto-Filho, A., Schroder, R. and Moreira, J.C. (2008) Xanthine oxidasedependent ROS production mediates vitamin A pro-oxidant effects in cultured Sertoli cells. Free Radic. Res. 42, 593–601. [32] Segel, H. (1986) Biochemical Calculations, 2nd ed, John Wiley and Sons Inc., New York. [33] Berg, J.M., Tymoczko, J.L. and Stryer, L. (2007) Biochemistry (sixth edition), W.H. Freeman and Company, New York. [34] Lin, C.T., Robertson, D.E., Ahmad, M., Raibekas, A.A., Jorns, M.S., Dutton, P.L. and Cashmore, A.R. (1995) Association of flavin adenine-dinucleotide with the Arabidopsis blue-light receptor Cry1. Science 269, 968–970. [35] Sokolowsky, K. et al. (2010) Spectroscopic and thermodynamic comparisons of Escherichia coli DNA photolyase and Vibrio cholerae Cryptochrome 1. J. Phys. Chem. B 114, 7121–7130. [36] Zhou, Z.M. and Swenson, R.P. (1996) The cumulative electrostatic effect of aromatic stacking interactions and the negative electrostatic environment of the flavin mononucleotide binding site is a major determinant of the reduction potential for the flavodoxin from Desulfovibrio vulgaris [Hildenborough]. Biochemistry 35, 15980–15988. [37] Bhattacharyya, S., Stankovich, M.T., Truhlar, D.G. and Gao, J.L. (2007) Combined quantum mechanical and molecular mechanical simulations of one- and two-electron reduction potentials of flavin cofactor in water, medium-chain acyl-CoA dehydrogenase, and cholesterol oxidase. J. Phys. Chem. A 111, 5729–5742. [38] Chang, F.C., Bradley, L.H. and Swenson, R.P. (2001) Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin. Biochimica Et Biophysica Acta-Bioenergetics 1504, 319–328. [39] Nozaki, D., Iwata, T., Ishikawa, T., Todo, T., Tokutomi, S. and Kandori, H. (2004) Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3. Biochemistry 43, 8373–8379. [40] Takahashi, Y. and Ebrey, T.G. (2003) Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment. Biochemistry 42, 6025–6034. [41] Nash, A.I., Ko, W.H., Harper, S.M. and Gardner, K.H. (2008) A conserved glutamine plays a central role in LOV domain signal transmission and its duration. Biochemistry 47, 13842–13849. [42] Jones, M.A., Feeney, K.A., Kelly, S.M. and Christie, J.M. (2007) Mutational analysis of phototropin 1 provides insights into the mechanism underlying LOV2 signal transmission. J. Biol. Chem. 282, 6405–6414. [43] Hendrischk, A.K. et al. (2009) A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides. Mol. Microbiol. 74, 990–1003. [44] Zirak, P., Penzkofer, A., Moldt, J., Pokorny, R., Batschauer, A. and Essen, L.O. (2009) Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana. J. Photochem. Photobiol. B-Biol. 97, 94–108. [45] Zeugner, A., Byrdin, M., Bouly, J.P., Bakrim, N., Giovani, B., Brettel, K. and Ahmad, M. (2005) Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function. J. Biol. Chem. 280, 19437– 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. (2009) Roles of NAD(+)/NADH and NADP(+)/NADPH in cell death. Curr. Pharm. Des. 15, 12–19. [47] Lanzl, K., von Sanden-Flohe, M., Kutta, R.J. and Dick, B. (2010) Photoreaction of mutated LOV photoreceptor domains from Chlamydomonas reinhardtii with aliphatic mercaptans: implications for the mechanism of wild type LOV. Phys. Chem. Chem. Phys. 12, 6594–6604. [48] Kim, S.K., Mason, J.T., Knaff, D.B., Bauer, C.E. and Setterdahl, A.T. (2006) Redox properties of the Rhodobacter sphaeroides transcriptional regulatory proteins PpsR and AppA. Photosynth. Res. 89, 89–98.
[49] Miller, G. (2006) Optogenetics: shining new light on neural circuits. Science 314, 1674–1676. [50] Ernst, O.P., Murcia, P.A.S., Daldrop, P., Tsunoda, S.P., Kateriya, S. and Hegemann, P. (2008) Photoactivation of channel rhodopsin. J. Biol. Chem. 283, 1637–1643. [51] Moglich, A., Ayers, R.A. and Moffat, K. (2010) Addition at the molecular level: signal integration in designed Per-ARNT-Sim receptor proteins. J. Mol. Biol. 400, 477–486.