Energetics of the H-Bond Network in Exiguobacterium sibiricum Rhodopsin.

Exiguobacterium sibiricum rhodopsin (ESR) functions as a light-driven proton pump utilizing Lys96 for proton uptake and maintaining its activity over a wide pH range. Using a combination of methodologies including the linear Poisson-Boltzmann equation and a quantum mechanical/molecular mechanical approach with a polarizable continuum model, we explore the microscopic mechanisms underlying its pumping activity. Lys96, the primary proton uptake site, remains deprotonated owing to the loss of solvation in the ESR protein environment. Asp85, serving as a proton acceptor group for Lys96, does not form a low-barrier H-bond with His57. Instead, deprotonated Asp85 forms a salt-bridge with protonated His57, and the proton is predominantly located at the His57 moiety. Glu214, the only acidic residue at the end of the H-bond network exhibits a pKa value of ∼6, slightly elevated due to solvation loss. It seems likely that the H-bond network [Asp85···His57···H2O···Glu214] serves as a proton-conducting pathway toward the protein bulk surface.

Exploring the Deprotonation Process during Incorporation of a Ligand Water Molecule at the Dangling Mn Site in Photosystem II.

The Mn4CaO5 cluster, featuring four ligand water molecules (W1 to W4), serves as the water-splitting site in photosystem II (PSII). X-ray free electron laser (XFEL) structures exhibit an additional oxygen site (O6) adjacent to the O5 site in the fourth lowest oxidation state, S3, forming Mn4CaO6. Here, we investigate the mechanism of the second water ligand molecule at the dangling Mn (W2) as a potential incorporating species, using a quantum mechanical/molecular mechanical (QM/MM) approach. Previous QM/MM calculations demonstrated that W1 releases two protons through a low-barrier H-bond toward D1-Asp61 and subsequently releases an electron during the S2 to S3 transition, resulting in O•- at W1 and protonated D1-Asp61. During the process of Mn4CaO6 formation, O•-, rather than H2O or OH-, best reproduced the O5···O6 distance. Although the catalytic cluster with O•- at O6 is more stable than that with O•- at W1 in S3, it does not occur spontaneously due to the significantly uphill deprotonation process. Assuming O•- at W2 incorporates into the O6 site, an exergonic conversion from Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (equivalent to the open-cubane S2 valence state) to Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (equivalent to the closed-cubane S2 valence state) occurs. These findings provide energetic insights into the deprotonation and structural conversion events required for the Mn4CaO6 formation.

Modulation of Electron Transfer Branches by Atrazine and Triazine Herbicides in Photosynthetic Reaction Centers.

Quinone analogue molecules, functioning as herbicides, bind to the secondary quinone site, QB, in type-II photosynthetic reaction centers, including those from purple bacteria (PbRC). Here, we investigated the impact of herbicide binding on electron transfer branches, using herbicide-bound PbRC crystal structures and employing the linear Poisson-Boltzmann equation. In contrast to urea and phenolic herbicides [Fufezan, C. Biochemistry 2005, 44, 12780-12789], binding of atrazine and triazine did not cause significant changes in the redox-potential (Em) values of the primary quinone (QA) in these crystal structures. However, a slight Em difference at the bacteriopheophytin in the electron transfer inactive branch (HM) was observed between the S(-)- and R(+)-triazine-bound PbRC structures. This discrepancy is linked to variations in the protonation pattern of the tightly coupled Glu-L212 and Glu-H177 pairs, crucial components of the proton uptake pathway in native PbRC. These findings suggest the existence of a QB-mediated link between the electron transfer inactive HM and the proton uptake pathway in PbRCs.

Interplay of two low-barrier hydrogen bonds in long-distance proton-coupled electron transfer for water oxidation.

D1-Tyr161 (TyrZ) forms a low-barrier H-bond with D1-His190 and functions as a redox-active group in photosystem II. When oxidized to the radical form (TyrZ-O•), it accepts an electron from the oxygen-evolving Mn4CaO5 cluster, facilitating an increase in the oxidation state (Sn; n = 0-3). In this study, we investigated the mechanism of how TyrZ-O• drives proton-coupled electron transfer during the S2 to S3 transition using a quantum mechanical/molecular mechanical approach. In response to TyrZ-O• formation and subsequent loss of the low-barrier H-bond, the ligand water molecule at the Ca2+ site (W4) reorients away from TyrZ and donates an H-bond to D1-Glu189 at Mn4 of Mn4CaO5 together with an adjacent water molecule. The H-bond donation to the Mn4CaO5 cluster triggers the release of the proton from the lowest pKa site (W1 at Mn4) along the W1…D1-Asp61 low-barrier H-bond, leading to protonation of D1-Asp61. The interplay of the two low-barrier H-bonds, involving the Ca2+ interface and forming the extended Grotthuss-like network [TyrZ…D1-His190]-[Mn4CaO5]-[W1…D1-Asp61], rather than the direct electrostatic interaction, is likely a basis of the apparent long-distance interaction (11.4 Å) between TyrZ-O• formation and D1-Asp61 protonation.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91out and W91in, with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91out conformation, the charge-separated state (FMN•-) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91in conformation. This leads to a lower activation energy for the charge separation in the W91out conformation, resulting in a faster charge separation compared to that in the W91in conformation. In the W91out conformation, the radical state (FMNH•) is more stable than FMN•- and forms from FMN•-, leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91in conformation, FMN•- cannot proceed further, returning to the dark-adapted state, as FMNH• is less stable. Thus, formation of the signaling state exclusively occurs in the W91out conformation.

Characterization of tryptophan oxidation affecting D1 degradation by FtsH in the photosystem II quality control of chloroplasts.

Photosynthesis is one of the most important reactions for sustaining our environment. Photosystem II (PSII) is the initial site of photosynthetic electron transfer by water oxidation. Light in excess, however, causes the simultaneous production of reactive oxygen species (ROS), leading to photo-oxidative damage in PSII. To maintain photosynthetic activity, the PSII reaction center protein D1, which is the primary target of unavoidable photo-oxidative damage, is efficiently degraded by FtsH protease. In PSII subunits, photo-oxidative modifications of several amino acids such as Trp have been indeed documented, whereas the linkage between such modifications and D1 degradation remains elusive. Here, we show that an oxidative post-translational modification of Trp residue at the N-terminal tail of D1 is correlated with D1 degradation by FtsH during high-light stress. We revealed that Arabidopsis mutant lacking FtsH2 had increased levels of oxidative Trp residues in D1, among which an N-terminal Trp-14 was distinctively localized in the stromal side. Further characterization of Trp-14 using chloroplast transformation in Chlamydomonas indicated that substitution of D1 Trp-14 to Phe, mimicking Trp oxidation enhanced FtsH-mediated D1 degradation under high light, although the substitution did not affect protein stability and PSII activity. Molecular dynamics simulation of PSII implies that both Trp-14 oxidation and Phe substitution cause fluctuation of D1 N-terminal tail. Furthermore, Trp-14 to Phe modification appeared to have an additive effect in the interaction between FtsH and PSII core in vivo. Together, our results suggest that the Trp oxidation at its N-terminus of D1 may be one of the key oxidations in the PSII repair, leading to processive degradation by FtsH.

Stretching vibrational frequencies and pKa differences in H-bond networks of protein environments.

The experimentally measured stretching vibrational frequencies of O-D [νO-D(donor)] and C=O [νC=O(donor)] H-bond donor groups can provide valuable information about the H-bonds in proteins. Here, using a quantum mechanical/molecular mechanical approach, the relationship between these vibrational frequencies and the difference in pKa values between H-bond donor and acceptor groups [ΔpKa(donor … acceptor)] in bacteriorhodopsin and photoactive yellow protein environments was investigated. The results show that νO-D(donor) is correlated with ΔpKa(donor … acceptor), regardless of the specific protein environment. νC=O(donor) is also correlated with ΔpKa(donor … acceptor), although the correlation is weak because the C=O bond does not have a proton. Importantly, the shifts in νO-D(donor) and νC=O(donor) are not caused by changes in pKa(donor) alone, but rather by changes in ΔpKa(donor … acceptor). Specifically, a decrease in ΔpKa(donor … acceptor) can lead to proton release from the H-bond donor group toward the acceptor group, resulting in shifts in the vibrational frequencies of the protein environment. These findings suggest that changes in the stretching vibrational frequencies, in particular νO-D(donor), can be used to monitor proton transfer in protein environments.

Absence of electron-transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center.

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes (Dods et al., (2021) Nature 589, 310-314), we investigated time-dependent changes in the energetics of the electron-transfer pathway, considering the entire protein environment of the protein structures and titrating the redox-active sites in the presence of all fully equilibrated titratable residues. In the dark and charge separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer-active branch (BL and HL) are higher than those in the electron-transfer-inactive branch (BM and HM). However, the stabilization of the charge-separated [PLPM]•+HL•- state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5 ps ([PLPM]•+HL•- state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL•- (saddling mode) is absent in the HL geometry of the original 5 ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron-transfer events in the XFEL structures.

Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex.

In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g = 2 multiline signal. However, its protonation state remains unclear. Here, we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g = 2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH-).

Structural and energetic insights into Mn-to-Fe substitution in the oxygen-evolving complex.

Manganese (Mn) serves as the catalytic center for water splitting in photosystem II (PSII), despite the abundance of iron (Fe) on earth. As a first step toward why Mn and not Fe is employed by Nature in the water oxidation catalyst, we investigated the Fe4CaO5 cluster in the PSII protein environment using a quantum mechanical/molecular mechanical (QM/MM) approach, assuming an equivalence between Mn(III/IV) and Fe(II/III). Substituting Mn with Fe resulted in the protonation of µ-oxo bridges at sites O2 and O3 by Arg357 and D1-His337, respectively. While the Mn4CaO5 cluster exhibits distinct open- and closed-cubane S2 conformations, the Fe4CaO5 cluster lacks this variability due to an equal spin distribution over sites Fe1 and Fe4. The absence of a low-barrier H-bond between a ligand water molecule (W1) and D1-Asp61 in the Fe4CaO5 cluster may underlie its incapability for ligand water deprotonation, highlighting the relevance of Mn in natural water splitting.

pH-Dependent Binding and Releasing Mechanism of Acetate in the Inner Water Cavity of Heliorhodopsin.

The high-resolution structure of heliorhodopsin crystallized at low pH reveals the presence of a planar triangle molecule, acetate, in the inner water cavity. Here, we investigate how the acetate molecule is stabilized at the counterion Glu107 moiety, using molecular dynamics (MD) simulations and a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations indicate that the density is best described as acetate among triangle acids, including nitric acid and bicarbonate. The calculated protonation state indicates that protonated acetate donates an H-bond to deprotonated Glu107 in the low-pH crystal structure. The observed red-shift of ∼30 nm in the absorption wavelength with pKa ≈ 4 is likely due to the His23/His80 protonation, rather than the Glu107 protonation. MD simulations also show that acetate can exist at the Glu107 moiety only when it is protonated. When ionized, acetate is released from the Glu107 moiety via Asn101 at the channel bottleneck and Arg91 on the intracellular protein surface. These observations could explain how acetate binds at low pH and releases at high pH.

Redox Potentials of Iron-Sulfur Clusters in Type I Photosynthetic Reaction Centers.

The electron transfer pathways in type I photosynthetic reaction centers, such as photosystem I (PSI) and reaction centers from green sulfur bacteria (GsbRC), are terminated by two Fe4S4 clusters, FA and FB. The protein structures are the basis of understanding how the protein electrostatic environment interacts with the Fe4S4 clusters and facilitates electron transfer. Using the protein structures, we calculated the redox potential (Em) values for FA and FB in PSI and GsbRC, solving the linear Poisson-Boltzmann equation. The FA-to-FB electron transfer is energetically downhill in the cyanobacterial PSI structure, while it is isoenergetic in the plant PSI structure. The discrepancy arises from differences in the electrostatic influences of conserved residues, including PsaC-Lys51 and PsaC-Arg52, located near FA. The FA-to-FB electron transfer is slightly downhill in the GsbRC structure. Em(FA) and Em(FB) exhibit similar levels upon isolation of the membrane-extrinsic PsaC and PscB subunits from the PSI and GsbRC reaction centers, respectively. The binding of the membrane-extrinsic subunit at the heterodimeric/homodimeric reaction center plays a key role in tuning Em(FA) and Em(FB).

Mechanism of Asparagine-Mediated Proton Transfer in Photosynthetic Reaction Centers.

In photosynthetic reaction centers from purple bacteria (PbRCs), light-induced charge separation leads to the reduction of the terminal electron acceptor quinone, QB. The reduction of QB to QB•- is followed by protonation via Asp-L213 and Ser-L223 in PbRC from Rhodobacter sphaeroides. However, Asp-L213 is replaced with nontitratable Asn-L222 and Asn-L213 in PbRCs from Thermochromatium tepidum and Blastochloris viridis, respectively. Here, we investigated the energetics of proton transfer along the asparagine-involved H-bond network using a quantum mechanical/molecular mechanical approach. The potential energy profile for the H-bond between H3O+ and the carbonyl O site of Asn-L222 shows that the proton is predominantly localized at the Asn-L222 moiety in the T. tepidum PbRC protein environment, easily forming the enol species. The release of the proton from the amide -NH2 site toward Ser-L232 via tautomerization suffers from the energy barrier. Upon reorientation of Asn-L222, the enol -OH site forms a short low-barrier H-bond with Ser-L232, facilitating protonation of QB•- in a Grotthuss-like mechanism. This is a basis of how asparagine or glutamine side chains function as acceptors/donors in proton transfer pathways.

Identification of the protonation and oxidation states of the oxygen-evolving complex in the low-dose X-ray crystal structure of photosystem II.

In photosystem II (PSII), the O3 and O4 sites of the Mn4CaO5 cluster form hydrogen bonds with D1-His337 and a water molecule (W539), respectively. The low-dose X-ray structure shows that these hydrogen bond distances differ between the two homogeneous monomer units (A and B) [Tanaka et al., J. Am Chem. Soc. 2017, 139, 1718]. We investigated the origin of the differences using a quantum mechanical/molecular mechanical (QM/MM) approach. QM/MM calculations show that the short O4-OW539 hydrogen bond (~2.5 Å) of the B monomer is reproduced when O4 is protonated in the S1 state. The short O3-NεHis337 hydrogen bond of the A monomer is due to the formation of a low-barrier hydrogen bond between O3 and doubly-protonated D1-His337 in the overreduced states (S-1 or S-2). It seems plausible that the oxidation state differs between the two monomer units in the crystal.

Substitution of Ca2+ and changes in the H-bond network near the oxygen-evolving complex of photosystem II.

Ca2+, which provides binding sites for ligand water molecules W3 and W4 in the Mn4CaO5 cluster, is a prerequisite for O2 evolution in photosystem II (PSII). We report structural changes in the H-bond network and the catalytic cluster itself upon the replacement of Ca2+ with other alkaline earth metals, using a quantum mechanical/molecular mechanical approach. The small radius of Mg2+ makes W3 donate an H-bond to D1-Glu189 in Mg2+-PSII. If an additional water molecule binds at the large surface of Ba2+, it donates H-bonds to D1-Glu189 and the ligand water molecule at the dangling Mn, altering the H-bond network. The potential energy profiles of the H-bond between D1-Tyr161 (TyrZ) and D1-His190 and the interconversion between the open- and closed-cubane S2 conformations remain substantially unaltered upon the replacement of Ca2+. Remarkably, the O5⋯Ca2+ distance is shortest among all O5⋯metal distances irrespective of the radius being larger than that of Mg2+. Furthermore, Ca2+ is the only alkaline earth metal that equalizes the O5⋯metal and O2⋯metal distances and facilitates the formation of the symmetric cubane structure.

Energetic Diversity in the Electron-Transfer Pathways of Type I Photosynthetic Reaction Centers.

Photosynthetic reaction centers from heliobacteria (HbRC) and green sulfur bacteria (GsbRC) are homodimeric proteins and share a common ancestor with photosystem I (PSI), classified as type I reaction centers. Using the HbRC crystal structure, we calculated the redox potential (Em) values in the electron-transfer branches, solving the linear Poisson-Boltzmann equation and considering the protonation states of all titratable sites in the entire protein-pigment complex. Em(A-1) for bacteriochlorophyll g at the secondary site in HbRC (-1157 mV) is as low as Em(A-1) for chlorophyll a in PSI (-1173 mV). Em(A0/HbRC) is at the same level as Em(A0/GsbRC) and is 200 mV higher than Em(A0/PSI) due to the replacement of PsaA-Trp697/PsaB-Trp677 in PSI with PshA-Arg554 in HbRC. In contrast, Em(FX) for the Fe4S4 cluster in HbRC (-420 mV) is significantly higher than Em(FX) in GsbRC (-719 mV) and PSI (-705 mV) due to the absence of acidic residues that correspond to PscA-Asp634 in GsbRC and PsaB-Asp575 in PSI. It seems likely that type I reaction centers have evolved, adopting (bacterio)chlorophylls suitable for their light environments while maintaining electron-transfer cascades.

Mechanism of Absorption Wavelength Shift Depending on the Protonation State of the Acrylate Group in Chlorophyll c.

Diatoms can use light in the blue-green region because they have chlorophyll c (Chlc) in light-harvesting antenna proteins, fucoxanthin and chlorophyll a/c-binding protein (FCP). Chlc has a protonatable acrylate group (-CH═CH-COOH/COO-) conjugated to the porphyrin ring. As the absorption wavelength of Chlc changes upon the protonation of the acrylate group, Chlc is a candidate component that is responsible for photoprotection in diatoms, which switches the FCP function between light-harvesting and energy-dissipation modes depending on the light intensity. Here, we investigate the mechanism by which the absorption wavelength of Chlc changes owing to the change in the protonation state of the acrylate group, using a quantum mechanical/molecular mechanical approach. The calculated absorption wavelength of the Soret band of protonated Chlc is ∼25 nm longer than that of deprotonated Chlc, which is due to the delocalization of the lowest (LUMO) and second lowest (LUMO+1) unoccupied molecular orbitals toward the acrylate group. These results suggest that in FCP, the decrease in pH on the lumenal side under high-light conditions leads to protonation of Chlc and thereby a red shift in the absorption wavelength.

Quantum mechanical analysis of excitation energy transfer couplings in photosystem II.

We evaluated excitation energy transfer (EET) coupling (J) between all pairs of chlorophylls (Chls) and pheophytins (Pheos) in the protein environment of photosystem II based on the time-dependent density functional theory with a quantum mechanical/molecular mechanics approach. In the reaction center, the EET coupling between Chls PD1 and PD2 is weaker (|J(PD1/PD2)| = 79 cm-1), irrespective of a short edge-to-edge distance of 3.6 Å (Mg-to-Mg distance of 8.1 Å), than the couplings between PD1 and the accessory ChlD1 (|J(PD1/ChlD2)| = 104 cm-1) and between PD2 and ChlD2 (|J(PD2/ChlD1)| = 101 cm-1), suggesting that PD1 and PD2 are two monomeric Chls rather than a "special pair". There exist strongly coupled Chl pairs (|J| > âˆ¼100 cm-1) in the CP47 and CP43 core antennas, which may be candidates for the red-shifted Chls observed in spectroscopic studies. In CP47 and CP43, Chls ligated to CP47-His26 and CP43-His56, which are located in the middle layer of the thylakoid membrane, play a role in the "hub" that mediates the EET from the lumenal to stromal layers. In the stromal layer, Chls ligated to CP47-His466, CP43-His441, and CP43-His444 mediate the EET from CP47 to ChlD2/PheoD2 and from CP43 to ChlD1/PheoD1 in the reaction center. Thus, the excitation energy from both CP47 and CP43 can always be utilized for the charge-separation reaction in the reaction center.

Electron-Transfer Route in the Early Oxidation States of the Mn4CaO5 Cluster in Photosystem II.

The electron transfer from the oxygen-evolving Mn4CaO5 cluster to the electron acceptor D1-Tyr161 (TyrZ) is a prerequisite for water oxidation and O2 evolution. Here, we analyzed the electronic coupling in the rate-limiting electron-transfer transitions using a combined quantum mechanical/molecular mechanical/polarizable continuum model approach. In the S0 to S1 transition, the electronic coupling between the electron-donor Mn3(III) and TyrZ is small (2 meV). In contrast, the electronic coupling between the dangling Mn4(III) and TyrZ is significantly large (172 meV), which suggests that the electron transfer proceeds from Mn3(III) to TyrZ via Mn4(III). In the S1 to S2 transition, the electronic coupling between Mn4(III) and TyrZ is also larger (124 meV) than that between Mn1(III) and TyrZ (1 meV), which favors the formation of the open-cubane S2 conformation with Mn4(IV) over the formation of the closed-cubane S2 conformation with Mn1(IV). In the S0 to S1 and S1 to S2 transitions, the Mn4 d-orbital and the TyrZ π-orbital are hybridized via D1-Asp170, which suggests that D1-Asp170 commonly provides a dominant electron-transfer route.

ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.