Coupling Drosophila Melanogaster Cryptochrome Light Activation and

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Coupling Drosophila Melanogaster Cryptochrome Light Activation and Oxidation of the Kv# Subunit Hyperkinetic NADPH Cofactor Gongyi Hong, Ruth Pachter, and Thorsten Ritz J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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The Journal of Physical Chemistry

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Coupling Drosophila Melanogaster Cryptochrome Light Activation and Oxidation of the Kvβ Subunit Hyperkinetic NADPH Cofactor Gongyi Hong,a Ruth Pachter,a* and Thorsten Ritzb a b

*

Air Force Research Laboratory, Wright-Patterson Air Force Base, OH 45433-7702

Department of Physics and Astronomy, University of California, Irvine, CA 92697-4575

Corresponding author: [email protected], Tel: (937) 255-9689 1 Environment ACS Paragon Plus

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ABSTRACT Motivated by observations on the involvement of light-induced processes in the Drosophila melanogaster cryptochrome (DmCry) regulation of the neuronal firing rate, which is achieved by a redox state change of its voltage-dependent K+ channel Kvβ subunit Hyperkinetic (Hk) NADPH cofactor, we propose in this work two hypothetical pathways that potentially may enable such coupling. In the first pathway, triggered by blue-light-induced formation of a radical-pair [FAD•−TRP•+] in DmCry, the hole (TRP•+) may hop to Hk, e.g. through a tryptophan chain, and oxidize NADPH, possibly leading to inhibition of the N-terminus inactivation in the K+ channel. In a second possible pathway, DmCry’s FAD•− is re-oxidized by molecular oxygen, producing H2O2, which then diffuses to Hk and oxidizes NADPH. In this work, by applying a combination of quantum and empirical-based methods for free energy calculations, we find that the oxidation of NADPH by TRP•+ or by H2O2, and the re-oxidation of FAD•− by O2 are thermodynamically feasible. Our results may have implication in identifying a magnetic sensing signal transduction pathway, specifically upon Drosophila’s Hk NADPH cofactor oxidation, with subsequent inhibition of the K+ channel N-terminus inactivation gate, permitting K+ flux.

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1.

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Introduction

To account for avian magnetoreception, a radical-pair generated in cryptochromes upon light activation, which was proposed by Schulten, Ritz and co-workers,1-2 has been drawing much interest, as recently reviewed3 (see references therein). Cryptochromes are blue-green light photoreceptors that perform a variety of light signaling functions in plants and have been shown to entrain the circadian rhythm in Drosophila melanogaster.4 Light absorption by the active co-factor FAD in cryptochromes leads to reduction of fully oxidized FADox to its semiquinone form via formation of FAD-TRP radicalpairs in a triad of three tryptophans, which is essentially conserved in all members of the cryptochrome family.5 This spin-correlated state is interconverted by hyperfine interactions with surrounding nuclei, and can be modulated by a weak Zeeman interaction that describes the alignment of the electron spins to the magnetic field. Further light-activated reduction to a doubly reduced FAD− form is possible. The original resting state of FADox is restored through slow re-oxidation in the dark. The formation of a FAD-TRP radical-pair during photoactivation is well established and multiple theoretical studies indicate that 50 T, i.e. earth’s magnetic field strength, can affect the reaction rates and/or yields of the radical-pair reaction steps in cryptochrome photoactivation.6-8 Experimentally, 1 mT magnetic field effects on cryptochrome radical-pairs have been observed in vitro.9 Further support for the cryptochrome hypothesis comes from behavioral studies with Drosophila melanogaster that have shown that cryptochrome (DmCry) is required for their light-dependent magnetoreception in T-mazes,10 and for magnetic field effects on the entrainment of their circadian rhythm.11 Likewise, behavioral and genetic studies with cockroaches, another invertebrate, demonstrate that magnetic orientation requires cryptochromes, which is consistent with involvement of all of the redox forms of flavin.10, 12-13 It has been speculated that a radical-pair formed during flavin re-oxidation in the dark may be involved in magnetic sensing.6, 14 Interestingly, Wiltschko et al.15 demonstrated by behavioral studies of Erithacus rubecula that magnetic sensing is possible in the dark interval when using intermittent light and magnetic field pulses, lending support to a possible magnetoreception role of a radical-pair formed during flavin re-oxidation. Evidence of dark re-oxidation by molecular oxygen of the cryptochrome in 3 Environment ACS Paragon Plus

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Arabidopsis thaliana (AtCry1) was reported by Müller and Ahmad,16 and further extended,17 accompanied by hydrogen peroxide and reactive oxygen species generation.18 While much attention has been given to the details of the formation of a putative magnetosensitive radical-pair in cryptochromes, signal transduction pathways for neuronal responses have not been widely investigated. One possibility is that cryptochromes might be close to their activation threshold and when the magnetic field triggers the cryptochrome light response, the usual light signal transduction pathways of cryptochromes are activated. The mechanism of cryptochrome light signal transduction is currently not yet fully understood and light induced conformational changes, phosphorylation and dimerization have all been suggested to play a role in the cryptochrome activation/deactivation mechanism. Downstream responses of cryptochrome activation would then lead to neuronal responses via as of yet unidentified pathways. Here, we explore the possibility that magnetic signaling is instead mediated through oxidation of the widely present metabolite NADPH (nicotine amide adenine dinucleotide phosphate). The rationale for a possible involvement of NADPH and linking cryptochromes to ion channels’ response, and hence neuronal responses, is as follows. First, we note that cryptochromes that respond to blue light regulate the neuronal firing rate in a subset of arousal and circadian neurons in Drosophila melanogaster.19-21 In this case, transduction is achieved by the redox sensor of the voltage-gated potassium channel β-subunit (Kvβ). Drosophila expresses a single Kvβ designated Hyperkinetic (Hk), based on a highly conserved aldo-keto-reductase (AKR) domain. Membrane depolarization depends on a redox change in DmCry but not on the conformational change in the DmCry C-terminal involved in circadian entrainment, and it is unclear how the redox coupling occurs.21 Light-dependent magnetic field modulation of action potential firing in Drosophila larvae motoneurons was also demonstrated,22 and it was shown that the effect of a 100 mT field on the seizure response of Drosophila larvae depends on the wavelength of the DmCry excitation.23 Notably, oxidation of the Kvβ-bound cofactor NADPH to NADP+ induces augmented channel activity,24 presumed to be due to the inhibition of the “ball and chain”-type inactivation.25 However, although it was previously indicated that there may be N-type inactivation to the targeted K+ 4 Environment ACS Paragon Plus


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channel via Hk,26 a proposed mechanism of “ball and chain” immobilization, as demonstrated for mammalian Kv1,25 has yet to be proven in this case. Nevertheless, these observations could imply that DmCry light activation possibly triggers biochemical processes that could lead, in part, to Drosophila’s Hk NADPH cofactor oxidation and subsequent ion channel response. In this work, we explored potential oxidants generated upon DmCry light excitation that could lead to oxidation of NADPH. In this context, note that reduction of FAD upon light activation results in an oxidized tryptophan residue (TRP•+), an oxidant, in addition to a FAD anion radical (FAD•−).9 The oxidant may trigger a chain of hole-hopping along a cascade of tryptophan residues, leading to NADPH oxidation. TRP/TYR chains were observed in glycoside hydrolases, which can transport potentially damaging oxidizing equivalents away from a fragile active site to the protein surface.27 We therefore considered the mechanism of coupling photo-activation of DmCry and oxidation of NADPH by TRP•+. In addition, we considered a second pathway that results from re-oxidation of reduced FAD by oxygen, followed by H2O2 production, which in turn may oxidize NADPH. Indeed, as noted, hydrogen peroxide and reactive oxygen species are generated during re-oxidation of FAD in AtCry1.18 More recently, accumulation of hydrogen peroxide and formation of superoxide resulting from the Drosophila cryptochrome photocycle was indicated.28 Re-oxidation of FAD•− in DmCry was reported,28 and the production of H2O2 proposed to occur in the following steps: (1) FAD•− + 3 O2 → FAD+2 O•− 2 ; (2) • • •− + H + + O•− 2 → HO2 ; (3) HO2 + O2 + H → H2 O2 + O2 . Effects of a weak magnetic field on in vivo

production of H2O2,29-30 and on the cellular H2O2/O2•− ratio, were also studied.31 Oxidation of NADPH by reactive oxygen species (ROS),32 and reversible oxidative regulation of voltage-gated K+ channels by ROS were investigated.33 We explored these two hypothetical pathways, which may couple the photo-induced redox reactions in DmCry to the oxidation of NADPH in Hk. While O2•− and singlet oxygen are short lived, H2O2 is more stable (cellular half-life~1ms34) and is freely exchangeable between intracellular compartments.35 To examine the thermodynamic feasibility of the proposed pathways, free energy calculations by a combination of quantum and free energy empirical molecular dynamics simulations were performed. 5 Environment ACS Paragon Plus

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We found that both pathways are thermodynamically feasible. Finally, the possible relevance of the results to transduction of magnetic sensing is discussed. 2.

Methods and Computational Details

2.1. Hk structure by homology modeling and MD refinement The 3D structure of the Drosophila Hk36 was built by homology modeling using the SWISSMODEL37 and I-TASSER38 methods with a homologous Kvβ2 X-ray structure39 (pdb:3EAU) as the template, shown in Figure 1(a). Note that the Kvβ2 is a β subunit that does not harbor an N-terminus that could function according to a ball-and-chain mechanism. Protein structure alignment between the Hk homologous structure and the template was carried out using TM-align,40 and the resulting TMscore of 0.94 and RMSD of 0.57 Å indicate the high similarity between the two structures (see Figure 1(b)). Hk shows almost the same fold as the template structure, including the location and orientation of secondary structures and the position of the NADPH cofactor.

(a)

(b)

Figure 1. (a) 3D structure of Hk built by homology modeling. (b) Alignment between the structures of Hk and Kvβ2. Colors for the (Kvβ2:Hk) secondary structures are: helix (purple:green), -sheet (cyan:yellow), coil or turn (pink:ice-blue); NADPH (red:blue).



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Molecular dynamics (MD) simulations of Hk at constant NVT (T=300K) for 500 ns using the AMBER14SB41 force field were then carried out using GROMACS version 5.0.x.,42 with appropriate force field parameters for NADPH,43 hydrogen peroxide,44 and molecular oxygen.45 Particle mesh Ewald sums46 were applied for efficient treatment of long-range electrostatics, with a short-range cutoff of 1 nm, and relative strength of the electrostatic interaction at the cut-off of 10-5 (spacing of 0.12 nm, interpolation order of 6). A 1.2 nm cut-off was used in calculating the Lennard-Jones potential. The LINCS algorithm was chosen to reset bonds to the correct lengths after an unconstrained update.47 The temperature was regulated by velocity rescaling with a stochastic term of 0.1 ps, and the pressure by the Berendsen weak coupling method. The protein was solvated in a box of water with a distance of 1 nm between the solute and the box wall. 2.2. Free energy MD simulations The free energy change from reactant (r) to product (p) is defined by ∆𝐺 = ∆𝐸 𝑄𝑀 [𝑟 → 𝑝] + ∆𝐺 𝑠𝑜𝑙 [𝑟 → 𝑝], where ∆𝐸 𝑄𝑀 [𝑟 → 𝑝] is calculated quantum mechanically and ∆𝐺 𝑠𝑜𝑙 [𝑟 → 𝑝] describes the solvation free energy difference, calculated by applying the linear response approximation.48 Specifically, for electron transfer, ΔG = 0.5(A + < VB-VA>B), where VA(VB) are the potential energies when the active site is in oxidation states A(B), and represents an average over sampling trajectories, obtained by all-atom MD simulations (for 0.5 ns). The MOLARIS program using the ENZYMIX force field49 were employed for the protein and explicit water. The simulated system (protein and solvent) is spherical and divided into four regions. Region I comprises the quantum mechanical active site, and region II the unconstrained protein atoms and explicit water molecules up to 24 Å from the center of region I, treated with ENZYMIX.49 Region III contains a 2 Å shell of Langevin dipoles that embed region II. Region IV is a dielectric continuum surrounding region III that accounts for bulk effects. The standard surface constrained all-atom solvent technique to account for the solvent50 and the local reaction field long-range treatment,51 were applied. 2.3. Electronic structure calculations


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When needed, the calculation of the energy of electron transfer was performed by the equallyweighted state-averaged over same spin multiplicity complete active space SCF approach (CASSCF)52 and extended multi-configuration quasi-degenerate perturbation theory (XMCQDPT2)53 based on MCQDPT,54 as implemented in Firefly.55 In the calculation of the energy of electron transfer from NADPH to TRP•+, the active space included HOMO of TRP•+ and HOMO of NADPH. DFT at the PBE026,27/6-31+G* level was also applied for geometry optimization and calculation of the energy of electron transfer, as appropriate. Frozen density functional theory (FDFT) calculations were also considered. Briefly, based on valence bond theory, for an electron transfer reaction, the reactant (r) and product (p) states can be described by valence bond like wave functions, such that for A+D→A− +D+, Ψr= Ψ[A]Ψ[D], Ψp=Ψ[A−]Ψ[D+]. FDFT, recently reviewed,56 is summarized in the following, using the notation in Ref.57 We start from the Kohn-Sham formalism for an N-electron system, i.e. 𝐸[𝜌] = 𝑇[𝜌] + ∫ 𝑉𝑒𝑥𝑡 (𝑟)𝜌(𝑟) 𝑑𝑟 + 1

∬ 2

𝜌(𝑟)𝜌(𝑟 ′ ) [𝑟−𝑟 ′ |

𝑑𝑟𝑑𝑟′ + 𝐸𝑥𝑐 [𝜌], where Vext is the external potential, Exc density functional of the exchange1

𝑁 ∗ ∗ 2 correlation energy, and 𝜌(𝑟) = ∑𝑁 𝑖=1 𝜑 (𝑟)𝜑(𝑟); 𝑇[𝜌(𝑟)] = ∑𝑖=1 𝜑 (𝑟) (− 𝛻 ) 𝜑(𝑟). The system of 2

interest is divided into subsystems. The Kohn-Sham formulation is applied to each subsystem, considering the interaction between them. In the case of two subsystems with 𝑁𝐼 and 𝑁𝐼′ electrons, the total

energy

of

the

system 1

𝐸[𝜌𝐼 , 𝜌𝐼′ ] = 𝑇[𝜌𝐼 ] + 𝑇[𝜌𝐼′ ] + ∫ 𝑉𝑒𝑥𝑡 (𝑟)𝜌(𝑟) 𝑑𝑟 + ∬ 2

𝜌(𝑟)𝜌(𝑟 ′ ) [𝑟−𝑟 ′ |

is

given

by

𝑑𝑟𝑑𝑟′ + 𝐸𝑥𝑐 [𝜌] + 𝑇𝑠𝑛𝑎𝑑𝑑 [𝜌𝐼 , 𝜌𝐼′ ],

where

∫ 𝜌𝐼 (𝑟) 𝑑𝑟 = 𝑁𝐼 , ∫ 𝜌𝐼′ (𝑟) 𝑑𝑟 = 𝑁𝐼′ , 𝜌(𝑟) = 𝜌𝐼 (𝑟) + 𝜌𝐼′ (𝑟) and 𝑇𝑠𝑛𝑎𝑑𝑑 [𝜌𝐼 , 𝜌𝐼′ ] = 𝑇𝑠 [𝜌]- 𝑇𝑠 [𝜌𝐼 ]−𝑇𝑠 [𝜌𝐼′ ], and Ts[ρ] is the density functional of the kinetic energy. When the exchange energy functional includes 𝑝𝑏𝑒0

Hartree-Fock exchange, e.g. as in PBE0, the exchange energy is given by 𝐸𝑥 [𝜌] = 𝐸𝑥 𝑝𝑏𝑒0

𝐸𝑥

[𝜌𝐼 ] +

[𝜌𝐼′ ] + (𝐸𝑥𝑝𝑏𝑒96 [𝜌𝐼 + 𝜌𝐼′ ]−𝐸𝑥𝑝𝑏𝑒96 [𝜌𝐼 ] − 𝐸𝑥𝑝𝑏𝑒96 [𝜌𝐼′ ]). FDFT calculations were performed using

a parallel C++ code developed locally. An improvement of a previous code58 also includes analytical evaluation of GTO 1e and 2e integrals using the McMurchie-Davidson formulation.59 An optimal 8 Environment ACS Paragon Plus


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implementation of the Coulomb matrix evaluation was achieved by avoiding the explicit evaluation of four-center two-electron integrals60 and two-center 2e integrals of an Hermitian Gaussian type function basis [p|q].61 To benchmark FDFT vs. DFT, calculation of the energy of electron transfer from NADP• to TRP•+ was performed for the [NADP … TRP] model system (see Figure S1). Energy differences between the triplet reactant 3[NADP• … TRP•+] and the singlet product 1[NADP+ … TRP] were less than 0.1 eV, with values of -2.48, -2.54 and -2.52 eV, at the DFT PBE0/6-31+G*, DFT PBE0/6-31G*, and FDFT PBE0/6-31G* levels, respectively, thus justifying use of the method. Total energies (PBE0/6-31G*) were -1200.6634 au (-1200.6557 au) for DFT (FDFT) calculations for the triplet reactant 3[NADP• … TRP•+] and -1200.7569 au (-1200.7483 au) for DFT (FDFT) for the singlet product 1[NADP+ … TRP]. 3. Results and Discussion 3.1. Oxidation of NADPH by TRP•+ First, we consider an Hk-DmCry complex that may facilitate TRP•+ hopping from DmCry to Hk. It is known62 that Kvβ proteins associate with T1 domains (T1) of Kv1 channels (T1 domains and Kvβ proteins form a T14β4 complex). Using a full length Kv1 channel X-ray structure (pdb:3LUT),63 we derived a T14Hk4 complex by protein structure alignment using TM-align.40 The protein-protein docking of T14Hk4 and DmCry (pdb:4GU5) was carried out using ZDOCK,64 as shown in Figure 2(a); there are potentially four binding sites for DmCry. Interestingly, a feasible chain of TRP residues that may enable hole-hopping from DmCry to Hk was found, including TRP420, TRP397, TRP342 and TRP394 in DmCry, and TRP349, TRP402, and TRP282 in Hk (see Figure 2(b)). The four tryptophan residues in DmCry coincide with the so-called tetra tryptophans, which were found essential for transport to the protein surface after photo-induced formation of a [FAD•− … TRP•+] radical-pair.65


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(a)

(b) Figure 2. (a) 3D structure of the T1-Hk-DmCry complex (left, top view; right, side view). (b) TRP chain for electron transfer from Hk to DmCry (left to right) or hole-hopping from DmCry to Hk. Next, NADPH oxidation by TRP•+402 was investigated. The quantum region is depicted in Figure 3(a). Geometry optimization was carried out at the PBE0/6-31+G* level, and calculation of the energy of electron transfer using XMCQDPT2/6-31G*, following a CASSCF calculation with 3 electrons and 2 active molecular orbitals, where the HOMO and HOMO-1 (shown in Figure 3(b)) were included. The reaction energy was calculated as -1.04 eV, -1.26 eV and -1.20 eV, using CASSCF, XMCQDPT2, and 𝑠𝑜𝑙 (𝑟 FDFT, respectively (see Table 1). The solvation free energy difference ∆𝐺𝑚𝑚 → 𝑝) was calculated as

described in section 2.2., resulting in a value of -0.16 eV. The final free energy of NADPH to NADPH+ oxidation by TRP•+402 is -1.42 eV. 10 Environment ACS Paragon Plus


(a)

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(b)

Figure 3. (a) Quantum region for calculation of the reaction free energy of electron transfer from NADPH to TRP•+402 in Hk. (b) Top two highest occupied molecular orbitals in the CASSCF calculation (HOMO: red and blue, HOMO-1: light blue and purple). Further oxidation of NADP• by TRP•+ was then considered. Geometry optimizations were carried out for both triplet reactant and singlet product at the PBE0/6-31+G* level, resulting in an electron transfer reaction energy of -1.98 eV (see Table 1). The structure of the NADP• model compound is given in Figure S2. We note that both NADP• and TRP•+402 have an unpaired electron, and spin coupling gives rise to either a triplet or a singlet, and after one electron transfer from NADP• to TRP•+402, the product is a closed-shell singlet (see Figure 4). Since direct conversion of a triplet reactant to a singlet product is spin-forbidden, we calculated the energy of the singlet reactant as follows. The wave functions of a system with two singly occupied molecular orbitals a and b are given by 31Ψ = |a+ b+ |, 3 0Ψ

3 −1Ψ

= |a− b− |,

= (|a+ b− | + |a− b+ |)/√2 for triplet states, and 10Ψ = (|a+ b− | − |a− b+ |)/√2 for a singlet state.

Three degenerate triplet states have the same energy E(RT), which can be calculated by DFT for the triplet 31Ψ, since it can be described by a single determinant. The energy of the reactant singlet E(RS) can be derived from (𝐸𝑇 [ 30𝛹 ] + 𝐸𝑆 [ 10𝛹 ])/2 = 𝐸[|𝑎+ 𝑏 − |], where a(b) is a singly occupied molecular orbital,66 located at NADP• (TRP•+402). The calculated energy difference between the singlet reactant and triplet reactant was -0.05 eV. Finally, the calculated solvation free energy difference between 𝑠𝑜𝑙 (𝑟 reactant and product (∆𝐺𝑚𝑚 → 𝑝)) was 0.06 eV. The calculated down-hill free energy change for


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oxidation of NADPH by TRP•+ is consistent with the reported redox reaction free energy change in solution. Measured potentials (vs. NHE) for TRP•+/TRP in solution of 1 V,67 for NADP•/NADPH of 0.28 V and NADP+/NADP• of -0.92 V,68 indicate a free energy change of -0.72 eV for oxidation of NADPH by TRP•+ and -1.92 eV for oxidation of NADP• by TRP•+.

Table 1. Free energy change of oxidation of NADPH by TRP•+402. NADPH+TRP•+→NADP•(H+)+TRP ΔE (CASSC/6-31G*) (r → p)

-1.04 eV

ΔE (FDFT/6-31G*) (r → p)

-1.20eV

ΔE (CASSC/XQCPT2/6-31G*) (r → p)

-1.26 eV

𝑠𝑜𝑙 (𝑟 ∆𝐺𝑚𝑚 → 𝑝)

-0.16 eV

∆𝐺 (𝑟 → 𝑝)

-1.42eV

NADP•+TRP•+→NADP++TRP ΔE (PBE0/6-31+G*) (𝑟𝑇 → 𝑝)

-1.98 eV

ΔE (PBE0/6-31+G*) (𝑟𝑆 → 𝑟𝑇 )

-0.05 eV

ΔE (PBE0/6-31+G*) (𝑟𝑠 → 𝑝)

-2.03 eV


0.06 eV

∆𝐺 (𝑟𝑆 → 𝑝)

-1.97 eV

a

NADPH+ has a low pKa of ca. -3.5,69 and we assume NADPH+ deprotonated.

Triplet reactant (RT)

Singlet ractant (RS)

Singlet product (Ps)

Figure 4. Frontier molecular orbitals in the oxidation of NADP• by TRP•+ in Hk.



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Alternatively, because NADP• can reduce molecular oxygen in aqueous solution (rate constant 2×109 M-1s-170), we also considered oxidation of NADP• by molecular oxygen rather than by a TRP•+. After exploration of possible oxygen binding sites that stabilize O2•−, the site near ARG348 and HIS318 was chosen for further investigation because of feasible stabilization by the positively charged residue. Geometry optimization of a model compound (shown in Figure 5) was performed by PBE0/6-31+G*, including all first shell neighboring residues and backbones around NADPH and O2.

Figure 5. Model compound derived from the region of ARG348 and HIS318 in Hk for calculation of the oxidation of NADP• by O2•. Since ground-state molecular oxygen is a triplet, the reactant can be either a quartet ( 4 [ 3 𝑂2 + 2

𝑁𝐴𝐷𝑃• ]) or a doublet ( 2 [ 3 𝑂2 + 2 NADP • ]), while the product can only be a doublet ( 2 [ 2 O•− 2 +

1

NADP + ]), where the spin charge is 1 for O•− 2 and 2 for ground-state O2. The calculated reaction energy

𝑠𝑜𝑙 (𝑟 was -0.20 eV. The calculated solvation free energy difference ∆𝐺𝑚𝑚 → 𝑝) was -0.92 eV by MD

simulations, where the protein dipole contributes 0.22 eV and the solvent dipole -1.14 eV. The final reaction free energy of the oxidation of NADP• by O2 is -1.12 eV. Our calculations show a similar thermodynamic trend to the measured redox potential in aqueous solution, namely of -0.92V68 for NADP+/NADP•, and -0.16 V71 for O2/O2•−, resulting in a reaction free energy of -0.76 eV. 13 Environment ACS Paragon Plus

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3.2. Oxidation of NADPH by H2O2 First, we discuss re-oxidation of reduced FAD (FAD•−). Following O2 positioning at binding sites near the FAD cofactor, MD simulations were performed for refinement. To explore configuration space, after 10 ns the O2/O2•− reduction potential at each site was estimated, including only O2 in the active site. Binding sites surrounding CYS110, HIS138, ARG271 and ARG315 were found most favorable for reduction of O2. Oxidation of FAD•− by O2 bound near ARG315 and ARG271 was further investigated using a model compound (see Figure 6), which includes first shell neighboring residues and backbones around FAD and O2. Spin multiplicities were as discussed above. The energy of electron transfer was 𝑠𝑜𝑙 (𝑟 0.30 eV, and the solvation free energy difference ∆𝐺𝑚𝑚 → 𝑝) -0.762 eV, where the protein dipole

contributes 1.86 eV and solvent dipole -2.62 eV. The final reaction free energy of the oxidation of FAD• by O2 was -0.46 eV.

Figure 6. Model compound for calculation of re-oxidation of FAD•− in DmCry, including first shell neighboring residues and backbones around FAD and O2. Oxidation of NADPH to NADP+ requires removal of two electrons and one proton, or one electron and one hydrogen atom, which can be realized by direct hydride transfer from NADPH to the oxidant. We define the reactant by [NADPH … H2O2] and the product by [NADP+ … H3O2−]. The model compound that includes all polar residues around H2O2 was based on a 500 ns MD equilibrated conformation, and the geometry was then optimized at the PBE0/6-31+G* level (see Figure 7). As 14 Environment ACS Paragon Plus


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illustrated in the product state, hydrogen peroxide converts into one water and one OH− after enacting a hydride. The reaction energy at the PBE0/6-31+G* level was -2.12 eV (see Table 2). The calculated 𝑠𝑜𝑙 (𝑟 solvation free energy difference (∆𝐺𝑚𝑚 → 𝑝)) was 0.52 eV.

Product

Reactant

Figure 7. Model compounds for oxidation of NADPH by H2O2 in Hk, including the polar residues around H2O2. The free energy perturbation approach72-73 was used to calculate the free energy difference between empirical

and

QM/MM

potentials

used

for

the

reactant

and

product

states,

namely

assuming exp{−𝛽∆𝐺𝑀𝑀→𝑄𝑀/𝑀𝑀 } =< exp{−𝛽[𝑉𝑄𝑀/𝑀𝑀 − 𝑉𝑀𝑀 ]} >𝑉𝑀𝑀 ; 𝛽 = 1/𝑘𝑇, where k is the Boltzmann constant and T the temperature. Sampling results are shown in Figure S3. The QM/MM correction to the solvation free energy difference was -0.13 eV. Thus, the final reaction free energy change for the oxidation of NADPH by H2O2 is -1.73 eV. The small QM/MM correction justifies omitting it in other free energy calculations in the systems we investigated. To calculate the solvation free energy difference for oxidation of NADPH by H2O2, the solvation free energy is defined as the free energy difference of two end states, where in state A the active site fully interacts with the environment, while in state B, only van der Waals interactions are turned on. This is achieved by a sequence of intermediate states introduced for the two end states (UA and UB), such that 𝑈(𝜆) = (1 − 𝜆)𝑈𝐴 + 𝜆𝑈𝐵


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48 and ∆𝐺 = 𝐺1 − 𝐺0 = ∑𝑛−1 𝑖=0 [𝐺𝜆𝑖+1 − 𝐺𝜆𝑖 ]. 𝐺𝜆𝑖+1 − 𝐺𝜆𝑖 , calculated using MOLARIS upon applying the

linear response approximation.

Table 2. Free energy change (in eV) of NADPH + H2O2→NADP+ + H2O + OH−. ΔE (PBE0/6-31+G*/gas phase) (𝑟 → 𝑝)

-2.12

𝑠𝑜𝑙 (𝑟 → 𝑝) ∆∆𝐺𝑚𝑚→𝑞𝑚/𝑚𝑚

-0.13


0.52

∆𝐺(𝑟 → 𝑝)

-1.73

3.3. Effects of the redox state of NADPH on the N-type inactivation of the K+ channel Kvβ1 and Kvβ2 have been shown to possess conserved cores with a functionally close to AKR that uses NADPH as a cofactor.62 Kvβ1 has an additional unstructured N-terminal segment of about 70 amino acids, which can restrain the channel inactivation by N-type blocking, where the N-terminus ARG37 and LYS139 bind GLU265 in the core domain, and ARG48 binds GLU349 when NADPH is oxidized to NADP+. This binding is loosened when NADP+ is reduced.25 Sequence alignment of Drosophila melanogaster Hk (AAC46631.1) with the rat Kvβ1 (NP_058999.1) shows that two positivenegative residue pairs are conserved in Hk, where (ARG165 and ARG163−GLU390) in Hk align with (ARG37, LYS39−GLU265) in Kvβ1, and (ARG180−GLU490) in Hk align with (ARG48−GLU349) in Kvβ1 (see Figure S4 for full alignment). It is thus reasonable to postulate a similar N-type inactivation upon oxidation of NADPH in Hk. However, the N-terminus in Hk is 128-residue longer than that in Kvβ1. In addition, four positively charged residues (LYS13, ARG15, ARG20, LYS24) in the Kvβ1 Nterminus, which were proposed to interact with the negatively charged residue in the T1 domain for Ntype inactivation74 are missing in Hk. 4. Conclusions In this work, we showed that the two-electron oxidation of NADPH to NADP+ in Hk by TRP•+, or one-electron oxidation by TRP•+ and by oxygen, are thermodynamically downhill. We found that 16 Environment ACS Paragon Plus


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optimal docking of DmCry to Hk results in a structure that allows hopping of a light-activated hole (TRP•+) to NADPH in Hk via a chain of tryptophan residues, potentially leading to NADPH oxidation. In addition, the formed [NADP• … TRP•+] radical-pair could contribute to magnetoreception. It is possible that 3O2, FAD•−, TRP•+ and NADP• can coexist at the same time. This finding may also explain the response under both blue and green light, since blue light induces reduction of FADox, while green light induces reduction of semi-reduced FAD,75 and both processes produce TRP•+. Our results may imply a possible pathway that couples the redox reaction in DmCry with that in Hk. Mutation of the relevant residues may hinder the hole-hopping chain. In addition, we found that the re-oxidation of reduced FAD species in DmCry and oxidation of NADPH by H2O2 in Hk is also thermodynamically downhill, suggesting that the second pathway is also possible. However, it was noted by Fogle et al.19 that both on and off response times when activating the circadian neurons in Drosophila melanogaster is about 0.1 s, which seems inconsistent with the second pathway since the FAD•− half-lifetime in vitro is 5.5 minutes28 and in vivo 15 minutes.76 A more complete kinetic description, beyond the scope of this study, may reconcile this second mechanism to experimental observations, e.g. by considering the competition of NADPH oxidation in Hk through H2O2 with scavenging of H2O2 by NADPH in the cytosol. Finally, our findings on favorable, potentially possible oxidation of NADPH, may imply an effect on the K+ channel and potentially a partial signal transduction mechanism. These results are consistent with recent observations on the coupling of light activation of DmCry to membrane depolarization by the K+ channel β-subunit redox sensor NADPH in Drosophila melanogaster.

Supporting Information Model system used in electron transfer calculations by FDFT vs. DFT (Figure S1). Structure of the NADP• model compound (Figure S2). Sampling in calculations of the reaction free energy of oxidation of NADPH by H2O2 (Figure S3). Sequence alignment of Hk and Kv1 (Figure S4). 17 Environment ACS Paragon Plus

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Coupling Drosophila Melanogaster Cryptochrome Light Activation and

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