Light-induced Hydrogen Bonding Pattern and Driving Force of Electron Transfer in AppA BLUF Domain Photoreceptor^,

Abstract

The AppA BLUF (blue light sensing using FAD) domain from Rhodobacter sphaeroides serves as a blue light-sensing photoreceptor. The charge separation process between Tyr-21 and flavin plays an important role in the light signaling state by transforming the dark state conformation to the light state one. By solving the linearized Poisson-Boltzmann equation, I calculated E_m for Tyr-21, flavin, and redox-active Trp-104 and revealed the electron transfer (ET) driving energy. Rotation of the Gln-63 side chain that converts protein conformation from the dark state to the light state is responsible for the decrease of 150 mV in E_m for Tyr-21, leading to the significantly larger ET driving energy in the light state conformation. The pK_a values of protonation for flavin anions are essentially the same in both dark and light state crystal structures. In contrast to the ET via Tyr-21, formation of the W state results in generation of only the dark state conformation (even if the initial conformation is in the light state); this could explain why Trp-104-mediated ET deactivates the light-sensing yield and why the activity of W104A mutant is similar to that of the light-adapted native BLUF.

Sensing blue light is a prerequisite for organisms to maintain their functions. BLUF2 domains serve as a photoreceptor by regulating the activity of covalently attached effector domains (1). The photoactivation of the BLUF domain from Rhodobactor sphaeroides is mediated by a red shift intermediate state that mainly consists of FAD and Tyr-21 (2). The electronic excited FAD^* state decays to the charge-separated Y-W- state. Via proton transfer (PT) from Y to , the Y-W- state transforms into the charge-neutral Y·-W-FADH· state and finally decays to the ground state (3). Recent spectroscopic studies suggested that not only Tyr-21 but also Trp-104 act as electron donors in the charge separation process, where Tyr-21 is the functionally relevant charge state (i.e. the productive pathway in Ref. 4), whereas formation of Trp-104 (i.e. the nonproductive pathway in Ref. 4) does not contribute to light sensing of AppA BLUF (4). Note that although FAD is the cofactor in the native BLUF domain (5), the BLUF fragment contains a mixture of flavins (i.e. FMN (majority), FAD, and riboflavin) when expressed in Escherichia coli (6). In fact, FMN is identified in the BLUF crystal structures from R. sphaeroides (7, 8). Currently, there are several points that need to be clarified for understanding the BLUF domain as described below.

In the protein environment of the BLUF domain, the orientation of the key residue Gln-63 with respect to Tyr-21 is a matter of debate. From observations of the native BLUF crystal structure, Anderson et al. (7) proposed that the –NH₂ group of the Gln-63 side chain forms a hydrogen bond with Tyr-21 in the dark state and that the –CO group of the Gln-63 side chain forms a hydrogen bond with Tyr-21 in the light state (Fig. 1). In contrast, from observations of the C20S mutant crystal structure, Jung et al. (8) assigned “dark” and “light” states that were opposite to those assigned by Anderson et al. (7). The majority of spectroscopic and mutational studies (9–12) suggested the same assignment of the dark and light structures as that proposed by Anderson et al. (7). In the present study, I tentatively follow the definition of Anderson et al. (7) if not otherwise specified (see Fig. 1 for the definition used in the present study).

FIGURE 1.

Changes in the orientation of the Gln-63 side chain from the dark state structure to the light state structure. Associated changes in the ET driving energy for the charge neutral [Y-W-FMN] (charge-separated [Y-W-] state are indicated in meV units.

Another open question is the functionally relevant location of Trp-104 with respect to the FMN binding site. In the crystal structure of the native BLUF domain refined by Anderson et al. (7), the indole nitrogen atom of the Trp-104 side chain is at a hydrogen-bonding distance from the carbonyl oxygen atom of the Gln-63 side chain (i.e. W_in conformation; see Fig. 2a). On the other hand, in the crystal structures of the C20S mutant by Jung et al. (8), Trp-104 is located on the protein surface and exposed to the bulk solvent (i.e. W_out conformation; see Fig. 2b). The W_out conformation is also identified in the BLUF domain from Thermosynechococcus elongatus (10). Jung et al. (8) argued that the W_in conformation reported by Anderson et al. (7) may be induced by the presence of detergent molecules. However, the W_in conformation can be seen in most NMR solution structural models of the BLUF domain (13). Recent spectroscopic studies suggested that Q63L and W104A mutants are insensitive to blue light, implying a functionally important role of a hydrogen bond between the two residues (that should exist only in the W_in conformation) (14). It is known that photoactivation of the BLUF domain induces the rotation of the Gln-63 side chain (7). However, the driving force of the Gln-63 rotation is yet unclear. It was proposed that proton movements (namely the PT from Y to the N5 atom of ) underlie the rotation from the observed kinetic isotope effects (3).

FIGURE 2.

Protein environment of the FMN binding site in the W_in (7) (Protein Data Bank code 1YRX) (a) and W_out (8) (Protein Data Bank code 2IYI) (b) structures in the dark state. Nitrogen and oxygen atoms are depicted as white and black balls, respectively.

To clarify the functional relevance of these possible four conformers (i.e. dark-W_out and light-W_out (Protein Data Bank 2IYI and 2IYG, respectively) (Fig. 1) (8), dark-W_in (Protein Data Bank 1YRX) (7), and light-W_in (no corresponding Protein Data Bank entry; see Fig. 3)), the values of the ET driving energy for each protein conformer that are yet experimentally unavailable need to be clarified.

FIGURE 3.

Dark and light state conformations of W_in (7) in transition from the charge-neutral [Y-W-FMN] state to the [Y-W-] state.

Recently, I calculated the redox (midpoint) potential (E_m) of flavin (15, 16) and redox-active tyrosine (17) precisely by considering the protonation states of all titratable sites in the proteins. In this study, I present the (E_m) of FMN for one-electron reduction (E_m(FMN/), protonated tyrosine for one-electron oxidation (E_m(Y/Y)), and tryptophan for one-electron oxidation (E_m(W/W)) in the BLUF domain, by solving the linearized Poisson-Boltzmann equation for all atoms in the crystal structures. From the calculated E_m, I obtain the driving energy of the ET in the charge separation process (via Tyr-21 and Trp-104) of the known four BLUF conformers. Then I evaluate (i) functional relevance of the conformers assigned by Anderson et al. (7) and Jung et al. (8), (ii) difference of the ET energetics in the W_in and W_out structures, and (iii) why the charge separation process involving Trp-104 does not lead to the light signaling state.

Since the dark and light state structures are originally available only for the W_out structure (8) (i.e. without performing the modeling of protein atomic coordinates), the computational results of the W_out structure (8) are mainly presented if not otherwise specified. In particular, the results of W_out and W_in were essentially the same in the ET via Tyr-21 but not in the ET via Trp-104. In the present study, I used the same conditions sufficiently evaluated in previous studies on proteins that contain flavin (15, 16) and redox-active tyrosine (17).

THEORY

Atomic Coordinates and Charges—For performing computations of the BLUF domain, the crystal structures of the native BLUF domains by Anderson et al. (7) (Protein Data Bank code 1YRX) and the C20S mutant BLUF domains by Jung et al. (8) (Protein Data Bank codes 2IYI and 2IYG) were used. For the W_out crystal structure by Jung et al. (8), I replaced the original S_γ atom of the C20 side chain with an oxygen atom and used it as the native BLUF structure. In this study, the crystal structures with Protein Data Bank code 2IYI and 2IYG were regarded as dark and light state structures (i.e. dark-W_out and light-W_out), respectively (Fig. 1). This definition of the dark and light structures is the same as that proposed Anderson et al. (7, 9–12).

For the W_in conformations, I used the crystal structure refined by Anderson et al. (7) as the dark state structure (i.e. dark-W_in). I modeled the light-W_in structure by rotating the side chain –NH₂ and –CO groups of Gln-63 by 180° along the C_γ-C_δ axis (Fig. 3). The obtained atomic coordinates of the Gln-63 side chain for the light-W_in structure in the chargeneutral [Y-W-FMN] state are shown in Table S1.

The positions of the hydrogen atoms were energetically optimized with CHARMM (18) by using the CHARMM22 force field. During this procedure, the positions of all nonhydrogen atoms were fixed, and the standard charge states of all of the titratable groups were maintained (i.e. basic and acidic groups were considered to be protonated and deprotonated, respectively). All of the other atoms whose coordinates were available in the crystal structure were not geometrically optimized. Note that the protein atomic coordinates obtained after the energy optimization process revealed that Tyr-21 is a hydrogen bond donor to the –NH₂ and –CO groups of the Gln-63 side chain in the dark and light structures, respectively, in agreement with what was observed in recent density functional theory studies (19).

Atomic partial charges of the amino acids were adopted from the all atom CHARMM22 (18) parameter set. Atomic charges of the 5′-phosphate group of FMN quinone (–H₃PO₃, , and ) were adopted from those of methylphosphate. The charges of FMN, and FMNH^· were from a previous report on flavodoxin (15). The atomic charges for the redox-active tyrosine were adopted from Ref. 20 (protonated tyrosine with neutral charge (Y) and protonated tyrosine radical with positive charge Y in the redox pair Y/Y). The atomic charges for the redox-active tryptophan were adopted from Ref. 20 (protonated tryptophan with neutral charge (W) and protonated tryptophan radical with positive charge W in the redox pair W/W).

Protonation Pattern, Redox Potential, and pK_a—The present computation is based on the electrostatic continuum model created by solving the linear Poisson-Boltzmann equation with the MEAD program (21). To facilitate a direct comparison with previous computational results, I uniformly used identical computational conditions and parameters, such as atomic partial charges and dielectric constants (e.g. see Refs. 15, 17, and 22). To obtain the absolute E_m values of the protein, we calculated the electrostatic energy difference between the two redox states in a reference model system using a known experimental E_m value. The difference in the E_m value of the protein relative to the reference system was added to the known E_m value (see below). All of the other titratable sites, including the 5′-phosphate group, were fully equilibrated to the redox state of FMN during the titration. The ensemble of the protonation patterns was sampled by the Monte Carlo method with Karlsberg.3 The dielectric constants were set to ε_p = 4 inside the protein and ε_w = 80 for water. All computations were performed at 300 K, pH 7.0, and an ionic strength of 100 mm. The linear Poisson-Boltzmann equation was solved using a three-step grid-focusing procedure at resolutions of 2.5, 1.0, and 0.3 Å. The Monte Carlo sampling yielded the probabilities [A_ox] and [A_red] of the two redox states of molecule A. E_m was evaluated using the Nernst equation. A bias potential was applied to obtain an equal amount of both redox states ([A_ox] = [A_red]), thereby yielding the redox midpoint potential E_m as the resulting bias potential. From this analogy, using the Henderson-Hasselbalch equation, pK_a can be calculated as the pH at which the concentrations of the protonated and deprotonated residue species are equal (Henderson-Hasselbalch pK_a). For convenience, the computed E_m value was given with mV accuracy, without implying that the last digit was significant. In general, an E_m value of ∼10 mV is a sufficiently reproducible range for the computational method used (e.g. see Refs. 15, 17, and 22).

pK_a and E_m Values in the Reference Model System— forms FMNH· upon protonation at the N5 nitrogen in FMN quinone (Fig. 1). The value of 8.6 (23) was taken as the pK_a(N5) value in the reference model system of equilibrium in aqueous solution. The value of 6.4 (24) was considered as the pK_a value of the 5′-phosphate group of . Note that the 5′-phosphate group was permanently deprotonated in the equilibrium (pK_a = 1.4 (24)) in all of the crystal structures that were investigated. Thus, in the present study, the equilibrium (pK_a = 6.4 (24)) was investigated, unless otherwise specified. As a reference model system, the following values for E_m versus the normal hydrogen electrode were used: E_m(Y/Y) = +1380 mV (25) and E_m(W/W) = +1070 mV (26) for one-electron oxidation in an aqueous solution. = -333 mV (15) was used as the reference model system of in an aqueous solution (see further discussion in the supplemental materials).

RESULTS AND DISCUSSION

Driving Energy in the ET Involving Tyr-21—By calculating and E_m(Y/Y), I obtained the driving energy (ΔG) of the ET between FMN and Y to be -156 and -317 meV for the dark and light state structures in the charge-separated Y-W- state, respectively (Table 1). I found that the calculated ΔG in the light state structure (-317 meV) is greater by 160 meV than that in the dark state structure (-156 meV; Table 1). This tendency essentially holds true for the chargeneutral [Y-W-FMN] state. Since the light state structure is energetically much favorable for the ET than the dark state structure in the present study, the charge separation process via Tyr-21 ([Y-W-FMN → Y-W- should complete with the light state structure (Fig. 1).

TABLE 1.

Redox potentials and ET driving energies in mV and meV units, respectively

Structure	Darka-Woutb(2IYIc)	Lighta-Woutb(2IYGc)	Darka-Winb(1YRXd)	Lighta-Winb(1YRXd(modeled)e)
Em(FMN/)
[Y-W-FMN]f	–564	–587	–556	–549
[Y-W-]g	–470	–496	–479	–425
[Y-W-]h	–473	–477	–454	NDi
Em(Y/Y)
[Y-W-FMN]f	1799	1645	1745	1683
[Y-W-]g	1724	1537	1702	1549
Em(W/W)
[Y-W-FMN]f	1145	1123	1334	1451
[Y-W-]h	1133	1116	1330	ND
ΔG (ET via Tyr-21)j
[Y-W-FMN]f	13	–118	–49	–118
[Y-W-]g	–156	–317	–169	–376
ΔG (ET via Trp-104)j
[Y-W-FMN]f	–641	–640	–460	–350
[Y-W-]h	–744	–757	–566	ND

^aDark (light) state structure where the –NH₂ (–CO) group of the Gln-63 side chain is a hydrogen-bonding partner of Tyr-21

^bW_out (W_in) conformation where Trp-104 is in (out of) the FMN binding pocket

^cSee Ref. 8

^dSee Ref. 7

^eThe –NH₂ and –CO groups of Gln-63 were rotated by 180° along the C_γ-C_δ axis, and their positions were energetically optimized with CHARMM (18)

^fE_m(FMN/), E_m(Y/Y), and E_m(W/W) were calculated in the charge-neutral (Y-W and W-FMN) states, respectively, by using the atomic coordinates of the ground [Y-W-FMN] conformer

^gE_m(FMN/) and E_m(Y/Y) were calculated in the charge-neutral (Y-W and W-FMN) states, respectively, by using the atomic coordinates of the [Y-W-] conformer. Hydrogen atom positions are energetically optimized in the presence of the [Y-W-] charge state

^hE_m(FMN/) and E_m(W/W) were calculated in the charge-neutral Y-W and Y-FMN states, respectively, by using the atomic coordinates of the [Y-W-] conformer. Hydrogen atom positions are energetically optimized in the presence of the [Y-W-] charge state

ⁱND, not determined

^jΔG (ET via Tyr-21) = E^m(Y/Y) – E^m(FMN/) – 2350 (meV), and ΔG (ET via Trp-104) = E^m(W/W) – E^m(FMN/) – 2350 (meV) (see, for instance, Ref. 3)

The revealed larger ET driving energy (i.e. energetically more exergonic) in the light state structure in the present study supports the validity of the assignment of the dark and light state conformations by Anderson et al. (7) and those suggested in spectroscopic and mutational studies (9–12).

Influence of Gln-63 on and E_m(Y/Y)—I investigated the influence of the BLUF protein environment on and E_m(Y/Y). The different orientations of the –NH₂ and –CO groups of Gln-63 in the dark and light structures alter E_m(Y/Y) by 230 mV (Table 2). Note that this influence is partially compensated by associated changes in the protonation states of titratable residues and the hydrogen atom coordinates of some residues, resulting in the net E_m shift of 154 mV (see Table 1). The lower E_m(Y/Y) in the light state structure implies that obviously, the proximity of the oxygen atom of the –CO group can stabilize the Y state effectively. In the dark state structure, the –CO group, in turn, destabilizes the Y state by 67 mV because of the proximity of its polar carbon atom to Tyr-21. On the other hand, the side chain orientation of Gln-63 does not essentially affect (Tables 1 and 3). Thus, the significant change in ΔG by switching from the dark state conformation to the light state conformation can be attributed predominantly to the change in E_m(Y/Y) and not that in .

TABLE 2.

Influence of the protein environment on E_m(Y/Y) for the [Y-W-FMN] ([Y-W-]) conformation in mV

	Em(Y/Y)
	Darka-Woutb(2IYI)c	Lighta-Woutb(2IYG)c
Redox potential
Em(p)d	1799	1645
Em(w)e	1380	1380
ΔEm(w→p)f	419	265
Protein element
Protein volumeg	341	339
Protein charge	78	–74
ΔPhosphateh	–14	–24
ΔBackbone	16	15
ΔSide chain	76	–65
Gln-63	102 (63)	–126 (–127)
–NH2	28 (–10)	–24 (–25)
–CO	67 (66)	–110 (–110)

^aDark (light) state structure where the –NH₂ (–CO) group of the Gln-63 side chain is a hydrogen-bonding partner of Tyr-21

^bConformation where Trp-104 is out of the FMN binding pocket

^cSee Ref. 8

^dE_m(Y/Y) in the BLUF domain

^eE_m(Y/Y) in water (25)

^fShift in E_m(Y/Y) from water to protein

^gInfluence of uncharged protein dielectric volume (i.e. the space covered by the merged van der Waals volumes of protein atoms)

^hInfluence of the FMN 5′-phosphate group. Note that this group is absent in FAD

TABLE 3.

Influence of the protein environment on E_m(FMN/) for the [Y-W-FMN] ([Y-W-]) conformation

	Em(FMN/)
	Darka-Woutb(2IYI)c	Lighta-Woutb(2IYG)c
Redox potential
Em(p)d	–564	–587
Em(w)e	–333	–333
ΔEm(w→p)f	–231	–254
Protein element
Protein volumeg	–207	–209
Protein charge	–24	–45
ΔPhosphateh	–56	–101
ΔBackbone	–20	–41
ΔSide chain	52	97
Tyr-21	8	2
Ser-23	–12 (–10)	–14 (–19)
Ser-41	–72 (–19)	–74 (–4)
Asn-45	–27	–20
–NH2	0	16
–CO	–25	–32
Gln-63	–2	0
–NH2	26	28
–CO	–27	–26

^aDark (light) state structure where the –NH₂ (–CO) group of the Gln-63 side chain is a hydrogen-bonding partner of Tyr-21

^bConformation where Trp-104 is out of the FMN binding pocket

^cSee Ref. 8

^dE_m(FMN/) in the BLUF domain

^eE_m(FMN/) in water (15)

^fShift in E_m(FMN/) from water to protein

^gInfluence of uncharged protein dielectric volume (i.e. the space covered by the merged van der Waals volumes of protein atoms)

^hInfluence of the FMN 5′-phosphate group. Note that this group is absent in FAD

Driving Force of Side Chain Rotation of Gln-63—Regardless of the difference in the hydrogen bond pattern with regard to the Gln-63 and FMN pair, the calculated pK_a(N5) values of FMN are essentially the same in both dark and light state structures (pK_a(N5) = 12.2 and 12.3 in the [Y-W-FMN] state and 14.0 and 13.9 in the [Y-W-] state, respectively). In other words, ΔG for the PT process from Y to is essentially the same in the dark and light state structures. On the other hand, as mentioned above, ΔG for the ET [Y-W-FMN → Y-W-] is 160 meV greater in the light state structure than that in the dark state structure ([Y-W-] state; Table 1). It is unlikely that the BLUF must maintain the dark state conformation during the charge separation process at the expense of this available 160-meV energy of the ET. Instead, it appears that the driving force of the Gln-63 side chain rotation is a result of the light-induced charge [Y-W-] (rather than the different ΔG for the PT from Y to the N5 atom of .

Indeed, the functional relationship between the rotation of a –CO-containing group and the light-induced charge can be seen in other photoassociated systems. In bacterial photosynthetic reaction centers from R. sphaeroides, light-induced charge separation leads to electron transfer from a monomeric bacteriochlorophyll to a bacteriopheophytin. It has been reported from spectroscopic studies that there exist two distinct conformations of the bacteriopheophytin in the reduced state with regard to the orientation of the acetyl group (27). The rotation of the acetyl group of the electron acceptor bacteriopheophytin takes place in coupling with the charge separation process after the excitation of chromophores (completed in 4 ps) (27). As a consequence, the rotation of the acetyl group contributes to a stabilization of the reduced state of the bacteriopheophytin by upshifting its E_m (28). Notably, this “light-induced acetyl conformational switch” can be activated by induced anionic charges on the bacteriopheophytin upon its photoreduction, leading to the stabilization of the charge-separated state in the photosynthetic reaction centers (27).

In analogy, the light-induced charge of the [Y-W-] state (rather than protonation process) may be the driving force for the rotation of the Gln-63 side chain; the Gln-63 reorientation is probably synchronized with the completion of the charge separation process [Y-W-FMN → Y-W-].

Driving Energy in the ET Involving Trp-104—In Ref. 3, the driving energy for the ET via Tyr-21 and Trp-104 was formerly estimated to be -620 and -400 meV, respectively, and it was concluded that Tyr-21 had a better signal overlap with flavin than Trp-104 due to the larger driving energy (3). On the other hand, the present study shows that the driving energy for the ET involving Trp-104 is significantly larger than that involving Tyr-21 (Table 1). Hence, the magnitude of the driving energy for the ET (via Tyr-21 and Trp-104) appears not to be a main factor that determines the ability of the light-signaling state formation of the BLUF domain (see below).

ET Driving Energy and Coupling in the W_in and W_out Conformations—There have been two different conformations identified in the crystal structures with regard to the location of Trp-104 (i.e. W_in (7) and W_out (8)) (Fig. 2). Their functional relevance is yet unclear experimentally (see discussions in Refs. 8 and 13). I found that the driving energy for the ET via Trp-104 in the dark-W_out (8) structure is 180 meV greater than in the dark-W_in (7) structure (Table 1). In contrast, the donor-acceptor distances (e.g. N_Trp-104-O4_FMN) are 3.8 and 14.5 Å for the W_in (7) and W_out (8) structures, respectively (see also Fig. 2). Obviously, the ET coupling of the W_in structure is significantly larger than that of the W_out structure (see Refs 30 and 31). Note that the corresponding distance for the ET via Tyr-21 (i.e. O_Tyr-21-N5_FMN) is 4.4–4.6 Å (7, 8), at the same level as the donor-acceptor distance for the ET via Trp-104 in the W_in structure.

Recent studies on Y21F and W104F mutants suggested that ET via Tyr-21 and Trp-104 compete in the charge separation process (4). Hence, if the dark-W_out structure is the only functionally relevant conformation, ET via Trp-104 may not be able to compete with ET via Tyr-21 due to its very low ET coupling. Thus, the W_in structure is probably necessary to explain the competition of the two ET processes (see below for further discussion).

Transformation of the Light-W_in Conformation to the Dark-W_in Conformation—In the W_in crystal structure, only the atomic coordinates of the dark state conformation (where the –NH₂ group of Gln-63 forms a hydrogen bond with the Tyr-21) are available (7). Therefore, to investigate the light-W_in conformation, I modeled the atomic coordinates by rotating the side chain –NH₂ and –CO groups of the Gln-63 side chain by 180° along the C_γ-C_δ axis and energetically optimizing the atomic coordinates of the Gln-63 side chain. In the [Y-W-FMN] state, E_m(W/W) of the dark-W_in structure was lower by ∼120 mV than that of the modeled light-W_in structure (Table 1), indicating that W can be stabilized effectively in the dark state structure. Note that the significantly destabilized W state in the modeled light-W_in structure is a result of the loss of the hydrogen bond between the indole nitrogen atom of Trp-104 with the –CO group of Gln-63 (Table 4).

TABLE 4.

Influence of the protein environment on E_m(W/W) for the [Y-W-FMN] ([Y-W-]) state conformation in mV

	Em(W/W)
	Darka-Winb(1YRX)c	Lighta-Winb(1YRX)c(modeled)d
Redox potential
Em(p)e	1334	1451
Em(w)f	1070	1070
ΔEm(w→p)g	264	381
Protein element
Protein volumeh	289	287
Protein charge	–25	94
ΔPhosphatei	–12	–12
ΔBackbone	18	15
ΔSide chain
Asn-45	102 (89)	99 (NDj)
–NH2	49 (37)	47 (ND)
–CO	49 (49)	48 (ND)
Gln-63	–74 (–75)	–12 (ND)
–NH2	–13 (–14)	0 (ND)
–CO	–61 (–61)	13 (ND)

^aDark (light) state structure where the –NH₂ (–CO) group of the Gln-63 side chain is a hydrogen-bonding partner of Tyr-21

^bConformation where Trp-104 is in the FMN binding pocket

^cSee Ref. 7

^dThe –NH₂ and –CO groups of Gln-63 were rotated by 180° along the C_γ-C_δ axis, and their positions were energetically optimized with CHARMM (18)

^eE_m(W/W) in the BLUF domain

^fE_m(W/W) in water (26)

^gShift in E_m(W/W) from water to protein

^hInfluence of uncharged protein dielectric volume (i.e. the space covered by the merged van der Waals volumes of protein atoms)

ⁱInfluence of the FMN 5′-phosphate group. Note that this group is absent in FAD

^jND, not determined (due to the irrelevant energetics of the protein structure)

By using the same procedure, I tried to generate the modeled light-W_in structure for the charge-separated [Y-W-] state. Unexpectedly, I could obtain only the dark state conformation for the [Y-W-] state and not the light state conformation (Fig. 3). In other words, when I apply the [Y-W-] charge state to the light state structure and then perform energy optimization of the atomic coordinates, the Gln-63 side chain undergoes a 180° rotation along the C_γ-C_δ axis, resulting in the atomic coordinates of the dark state structure during the energy optimization process (Fig. 3).

Functional Relevance of the W_in Conformations—The present study demonstrates that the light-W_in conformation of the [Y-W-] state is unusually unstable due to its energetically unfavorable hydrogen bond pattern. Thus, it is likely that the involvement of Trp-104 in the W_in charge separation process would lead to the elimination of the light-W_in conformation and, as a consequence, a decrease in the yield of the light signaling state of the BLUF domain. Indeed, spectroscopic studies on the W104F mutant indicate its 1.5-fold increase in the quantum yield of signaling state formation with respect to the native BLUF (32), implying that Trp-104 in the native BLUF is likely to lower the quantum yield of signaling state formation. This is in agreement with the present result.

In addition, recent mutational studies have suggested that there exist two competing light-induced ET pathways in the BLUF domain: one ET pathway via Tyr-21 that can form the signaling state and another ET pathway via Trp-104 that leads to the deactivation of signaling state formation (4). From the present result, only the W_in conformation is able to yield the dark state structure upon the formation of the [Y-W-] state (Fig. 3). In the W_out structure, there does not exist such a strict constraint on the orientation of the Gln-63 side chain because of the isolation of Trp-104 from Gln-63 (14.5 Å). Thus, to explain competitions of ET via Tyr-21 and Trp-104 (4, 32), the existence of the W_in conformation (7) is functionally required, as implied in former studies (7, 12–14).

Remarkably, spectroscopic studies by Masuda et al. (14) demonstrated that (i) the W104A mutant is insensitive to blue light and (ii) its activity is similar to that of the light-adapted native BLUF. These two facts are indicative that the loss of Trp-104 in the native W_in structure does not result in the transformation to the dark state structure and that the BLUF domain is always adapted to the light state structure in the absence of Trp-104. Formation of W in the W_in structure could reset the Gln-63 orientation to the dark (i.e. ground) state so that the BLUF domain can again perform photosensing effectively.

CONCLUSION

The driving energy of the ET via Tyr-21 in the light state structure is greater by 160 meV than that in the dark state structure (Table 1 and Fig. 1); this suggests that assignments of the BLUF dark/light conformers by Anderson et al. (7) are more reasonable than those by Jung et al. (8) to explain the energetics of the photoinduced ET event.

The calculated pK_a values indicate that the driving energy for the PT from Y to is essentially the same in the dark and light state structures.

The driving energy of the ET via Trp-104 is larger than that via Tyr-21.

I observed that W_in transforms the light state to the dark state (Fig. 3). This is in contrast to the case where Y transforms the dark state to the light state. Hence, W_in may facilitate to reset the Gln-63 orientation to the dark (i.e. ground) state so that the BLUF domain can again perform photosensing effectively. These are probably the reasons why the W104A mutant is insensitive to blue light and its activity is similar to that of the light adapted native BLUF in spectroscopic studies (14).