Light-Driven Enzyme Catalysis: Ultrafast Mechanisms and Biochemical Implications

Abstract

Light-activated enzymes are an important class of biocatalysts in which light energy is directly converted into biochemical activity. In most cases the light absorbing group is the isoalloxazine ring of an embedded flavin cofactor and in general two types of mechanism are in operation depending on whether the excited chromophore directly participates in catalysis or where photoexcitation triggers conformational changes that modulate the activity of a downstream output partner. This review will summarize studies on DNA photolyase, fatty acid photodecarboxylase (FAP), the monooxygenase PqsL, and flavin-dependent ene-reductases, where flavin radicals generated by excitation are directly used in the reactions catalyzed by these enzymes, and the blue light using FAD (BLUF) and light oxygen voltage (LOV) domain photoreceptors where flavin excitation drives ultrafast structural changes that ultimately result in enzyme activation. Recent advances in methods such as time-resolved spectroscopy and structural imaging have enabled unprecedented insight into the ultrafast dynamics that underly the mechanism of light-activated enzymes, and here we highlight how understanding ultrafast protein dynamics not only provides valuable insights into natural phototransduction processes but also opens new avenues for enzyme engineering and consequent applications in fields such as optogenetics.

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Structures and oxidation states of the flavin chromophore.

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Electron hopping through the Trp triad in DNA photolyase. The electron transfer chain in E. coli DNA photolyase. W382 serves as the primary donor to reduce the flavin and the resulting positive charge is stabilized on W306 within 30 ps which then deprotonates in 200 ns. The figure was adapted from Lukacs et al. and was made with PyMOL, from 1DNP.pdb. In addition to the Trp triad, the antenna chromophore 5,10-methenyltetrahydrofolate (MTHF) is also shown.

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Photochemical events upon reduction of FAD* in D. melanogaster (6–4) photolyase. (A) Key residues and processes. (1) FAD is reduced and Asp397 and Arg368 respond immediately. (2) Asn403 reacts similarly fast and undergoes a slow phase of response up to 20 ps. (3) A delayed (from 1 ps) and complex motion of water molecules is completed at 20 ps. (4) Met408 undergoes a photoreaction from 1 to 20 ps. (5) Trp381 is oxidized at 300 ps, with structural changes evolving around it up to 100 μs. (B) The kinetics of the observed difference electron density (DED) at key positions are shown. For water features the electron density is averaged over positive DED > 2σ and for the amino acids over negative DED < 2σ (side chains only). The radius of integration was 2.5 Å. Wat1 corresponds to feature V and Wat2 to feature VI in Figure of Cellini et al. The kinetics for water and Asn403 are vertically offset. The figure was taken from Cellini et al.

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The CPD photolyase reaction mechanism revealed by time-resolved femtosecond crystallography. Structural data of the CPD photolyase from Methanosarcina maze (mmCPD) in complex with a synthetic thymine dimer. , (A) and (B) Excitation of FADH^– results in formation of the FADH^–* excited state in 3 ps in which the isoalloxazine ring undergoes “butterfly bending” which results in a rearrangement of a hydrogen bond network around the ring that includes amino acid residues and ordered water molecules. This is followed by relaxation of the isoalloxazine ring into the geometry of the FADH^● semiquinone product within 3 ns. (C) Key intermediates in the DNA repair reaction catalyzed by photolyase. Selected intermediates (cyan) of the repair process are overlaid with the structure of the dark state (gray) to illustrate structural changes during catalysis. T7 and T8 are the thymine-7 and thymine-8, the damaged 5′- and 3′-thymines of the CPD lesion in the DNA strand. TT refers to the two thymines together. The figure was made from figures in Christou et al. and Maestre-Reyna et al.

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X-ray structure of the FAP product complex. Data were obtained from crystals at 150 K and pH 8.5 and show positive electron density from the polder omit map contoured at 3.5 rmsd, close to C432 that is assigned to bicarbonate (HCO₃ ^–). Also shown are the alkyl product (Alk), CO₂, and the isoalloxazine ring, which adopts a bent conformation. The figure was made using PyMOL and is adapted from Figure 4D in Sorigué et al.

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Proposed photocycle for the C. variabilis FAP.

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PqsL reaction and proposed mechanism of photoexcitation. (A) PqsL catalyzes the hydroxylation of aromatic amines such as the conversion of 2-aminobenzoylacetate (2-ABA) to 2-hydroxylaminobenzoylacetate (2-HABA). (B) Proposed mechanism for the photoactivation of FAD to FADH₂ via the disproportionation of two neutral flavin semiquinone species.

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Enzymatic photoreduction of FMN to FMNsq ^– (FMN^*–). FMN bound to 12-oxophytodienoate reductase (OPR1) is reduced by photoinduced electron transfer to generate FMNsq^–. Subsequently, FMNsq^– generates an α-acyl radical which stereoselectively cyclizes to form the oxindole product. SET, single electron transfer. The figure was redrawn based on Black et al.

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The OaPAC dimer and flavin binding pocket. (A) The OaPAC dimer (PDB: 8QFE). FAD (yellow) and ATP (purple) are shown as spheres in the BLUF and AC domains, respectively. Residues substituted with unnatural amino acids include Y6, W90, F103, Y125, and F180. (B) Dark state of OaPAC with Q48 in the keto tautomer and W90 in the ‘OUT’ conformation. The Q48 NH₂ is shown hydrogen bonded to the flavin N5 but not the C4 = O. (C) Light state of OaPAC with Q48 rotated and in the enol tautomer, and with W90 adopting the ‘IN’ conformation. The Q48 enol OH is hydrogen bonded to the flavin C4 = O and accepts a hydrogen bond from the W90 indole NH. , The movement of W90 is based on the structures of OaPAC determined by serial femtosecond crystallography and cryotrapping (PDB: 8QFE and 8GFG). (D) W90 and F103 were replaced with AzPhe. These residues interact across the dimer interface at the tip of the central helix. (E) F180 was replaced by AzPhe. (F) Y125 was replaced by fluoro-Tyr analogs. Figures (A), (D), (E), and (F) were made using PyMOL and taken/adapted from Jewlikar et al.

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Schematic of the proposed mechanism for the photocycle in the BLUF Slr1694 photoreceptor. (A) The dark-adapted state. Following photoexcitation of the flavin, Tyr8 reduces the flavin to produce a charge-separated state (orange arrow in A), resulting in proton transfer from Tyr8 to the flavin via Gln50 (blue arrows in A) to produce the structure in B. After proton transfer, the Gln50 imidic acid tautomer rotates to form a hydrogen bond with the flavin C4 = O carbonyl, as illustrated by the rotation of Gln50 from B to C. After charge recombination corresponding to electron transfer from the flavin to Tyr8 (orange arrow in C), the proton transfers back from the flavin to Tyr8, again via Gln50 (blue arrows in C) to produce the structure in D, which is the purported light-adapted state with a glutamine imidic acid tautomer. The figure was taken from Goings et al.

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TRIR of wild-type OaPAC and the n-FY6 OaPAC variants. Spectra recorded at 1, 5, 20, 40, 200, 1000, and 3000 ps are shown in the figure. (A) Wild-type (WT), (B) 3-FY6, (C) 2,3-F₂Y6, (D) 3,5-F₂Y6, and (E) 2,3,5-F₃Y6. The figure was taken from Tolentino Collado et al.

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Schematic summary of the OaPAC photocycle and fluoroTyr experiments. The figure was adapted from Tolentino Collado et al.

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TRMPS spectra of the W90AzPhe OaPAC dark state. (A) Time resolved IR difference response of the azido mode following ultrafast excitation of isoalloxazine cofactor in OaPAC at the W90 position. (B) EADS of W90AzPhe obtained from globally fitting the time-resolved data in A to a sequential first order kinetics model with an initial state (black) two intermediate states (red, blue) which form on a ns−μs time scale, and subsequently relaxes to a final state (green). The final state is compared with the FTIR difference spectra (black dash line). All analyses used the Glotaran software package. The figure was taken from Jewlikar et al.

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Structure of MsLadC. (A) The flavin binding pocket in the dark state. During photoactivation C53 forms an adduct with the flavin C4a. (B) X-ray structure of dark MsLadC dimer (PDB: 8C05). The BLUF and DGC domains are cyan and gray, and the linker is brown. FAD is yellow and pyrophosphate/Mg²⁺ is colored by atom. (C) The structure predicted by AlphaFold which is thought to be the light activated state.

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MD simulations of Jα-helix unfolding: The evolution in secondary structure and hydrogen bonding interactions between the flavin and N482, N492, Q513, and N414 for postadduct formation simulation times of 0 (dark state AsLOV2, PDB: 2V1A), 1.15, 4.53, and 6.81 μs (light state, MD). The figure was taken from Iuliano et al.

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Structural dynamics of the AsLOV2 photocycle revealed by TRXL. The photocycle of AsLOV2 includes the G state, three intermediates (I1, I2, and P), and related time constants (WT: 682 μs and 10.6 ms, and I427V: 130 μs and 3.4 ms), as determined from the kinetic analysis of the scattering data. The optimal structures (I1 and I2) indicate that the structural changes within the A’α and Jα helices allow the exposure of the β-scaffold to the external environment. Subsequently, AsLOV2 undergoes dimerization (P), utilizing the dimeric interface formed between their β-scaffolds. The figure was reproduced from Kim et al.