Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme

Abstract

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases with their flavin chromophore (FAD) utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD •isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge promotes proton transfer from arginine to FAD •-. Our molecular movies demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

Femtosecond conformational changes in a photolyase guide long-range electron transfer as resolved by serial crystallography

“…Refined light structures indicate a transient distance increase across the salt bridge caused by rotation of Asp397 (Table 1). We did not detect a light induced interaction between Arg368 and the FAD, which was observed on microsecond time scales [34]. Since the response of the salt bridge is present at 400 fs and quickly decays, we conclude that it is an effect of the sudden change of electrostatics on the reduced FAD.…”

“…The technique has been used for deciphering structural mechanisms in photosynthetic, sensor, and transport proteins [28][29][30][31][32][33]. With respect to photolyases, a nanosecond time-resolved SFX study has been carried for the two reduction steps on a cis-syn cyclobutane pyrimidine dimer (CBD) photolyase [34], and we have recorded a crystallographic snapshot of the end state of the first photoreduction in a (6-4) photolyase at 100 ms [35]. Here, we capture structural snapshots for the (6-4) photolyase from Drosophila Melanogaster covering the earliest, femto-and picosecond time scales.…”

Influence of pump laser fluence on ultrafast structural changes in myoglobin

“…On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer 1 . However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore [2][3][4][5][6][7][8][9][10][11][12][13][14] . As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern 15 whether this experimental approach 16 allows valid inferences to be drawn vis-à-vis biologically relevant single-photon-induced reactions 17 .…”

“…In time-resolved pump-probe SFX experiments, microcrystals are delivered into the XFEL beam using mostly liquid jets and diffraction data are collected at distinct time-delays following a photoexciting pump laser flash. On the sub-ps to ns timescale, this approach has been used to study isomerization reactions in photoactive yellow protein (PYP) 3 , fluorescent protein 4 , various rhodopsins 5,6,8,9,11,13 and phytochrome 7 ; electron transfer reactions in a photosynthetic reaction center 10 and photolyase 12 ; photocarboxylation 17 and photodissociation 2 . In all cases, a very high pump laser fluence was used to maximize the light-induced difference electron density signal, 16 .…”

Understanding Cysteine Chemistry Using Conventional and Serial X-ray Protein Crystallography

“…Each can be used to initiate catalysis at a defined time across the many molecules in the crystal lattice followed by monitoring the structures of the protein at defined timepoints after catalysis begins. Systems that are sensitive to light are ideal candidates for photoinitiation followed by time-resolved crystallography and include photosystem II [ 12 , 67 , 76 ], fatty acid photodecarboxylase [ 11 ], and photolyase [ 77 ] as well as a number of photoproteins that are not enzymes [ 78 ]. However, enzymes whose reactions are initiated by light are rare.…”