Short-Range Electron Transfer in Reduced Flavodoxin: Ultrafast Nonequilibrium Dynamics Coupled with Protein Fluctuations

Abstract

Short-range electron transfer (ET) in proteins is an ultrafast process on the similar time scales as local protein-solvent fluctuation, and thus the two dynamics are coupled. Here we use semiquinone flavodoxin and systematically characterized the photoinduced redox cycle with 11 mutations of different aromatic electron donors (tryptophan and tyrosine) and local residues to change redox properties. We observed the forward and backward ET dynamics in a few picoseconds, strongly following a stretched behavior resulting from a coupling between local environment relaxations and these ET processes. We further observed the hot vibrational-state formation through charge recombination and the subsequent cooling dynamics also in a few picoseconds. Combined with the ET studies in oxidized flavodoxin, these results coherently reveal the evolution of the ET dynamics from single to stretched exponential behaviors and thus elucidate critical time scales for the coupling. The observed hot vibration-state formation is robust and should be considered in all photoinduced back ET processes in flavoproteins.

Figure 1.

(A) X-ray crystal structure of semiquinone D. vulgaris flavodoxin (PDB code 4XF2) with the active site highlighted. (B) Close-up view of the active site with hydration water molecules obtained from a 500-ps MD simulation snapshot. FMNH˙ (purple) is sandwiched between W60 (green) and Y98 (yellow) at 3.5 Å and 3.3 Å, respectively. Neighboring residues of D95 (teal) and G61 (orange) are also shown. (C) Generalized timelines of photoinduced ET dynamics and active-site solvation relaxations in flavodoxin. The active site relaxations start from ~1 ps. For oxidized flavodoxin, the photoinduced cyclic ET is ultrafast within ~1 ps and senses a frozen environment. For semiquinone flavodoxin, the cyclic ET is in a few picoseconds and couples with active-site motions.

Figure 2.

Steady-states spectra of semiquinone flavodoxin. The absorption spectra of WT (blue) and Y98W (red) and the emission spectrum of the ET-inert mutant W60F/Y98F at longer wavelengths. Note the long-tail absorption of Y98W due to the electron delocalization with a charge-transfer character. The pump wavelength and the gated fluorescence wavelengths are marked in arrows. The top bars represent the absorption ranges of the excited state, various intermediates and final product. The transient-absorption probe wavelengths are also marked by top arrows.

Figure 3.

(A) Normalized femtosecond-resolved fluorescence transients of WT flavodoxin and mutants gated at 725 nm. (B) Normalized femtosecond-resolved fluorescence transients of Y98F and W60F gated at 652, 670 and 725 nm. Note the faster dynamics at the blue side resulting from the mixing of solvation and ET reaction.

Figure 4.

Normalized femtosecond-resolved absorption transients of the single donor W60 (Y98R) probed from 800 to 525 nm. Inset A shows the gradual changes with the different probe wavelengths. Note the longer dynamics probed at 690–650 nm. Insets B and C show the deconvolution of the transients into constitutive species probed at 655 and 630 nm, respectively. The excited-state signal of FMNH˙* in inset C is relatively small and not shown.

Figure 5.

Normalized femtosecond-resolved absorption transients of the single Y98 donor (W60F) probed from 800 to 525 nm. Inset A shows the gradual changes of the dynamics with the different probe wavelengths. Note the longer dynamics probed at 690–640 nm. Insets B and C show the deconvolution of the transients into constitutive species probed at 650 and 630 nm, respectively.

Figure 6.

Normalized femtosecond-resolved absorption transients with dual donors, (A) W60/Y98 (WT), (B) W60/Y98 (D95N), (C) Y60/Y98 (W60Y) and (D) W60/W98 (Y98W). Note similar patterns with a longer dynamics in the probe region of 690–640 nm and the difference in timescales for these mutants.

Figure 7.

Complete photoinduced redox cycle coupled with active-site relaxations including hydration water molecules. Note the formation of vibrationally excited products FMNH˙^† after charge recombination that subsequently decay in 3–5 ps to complete the ET cycle.