Photoisomerization in Proteorhodopsin mutant D97N

Abstract

The first steps of the photocycle of the D97N mutant of proteorhodopsin (PR) have been investigated by means of ultrafast transient absorption spectroscopy. A comparison with the primary dynamics of native PR and D85N mutant of bacteriorhodopsin is given. Upon photoexcitation of the covalently bound all-trans retinal the excited state decays biexponentially with time constants of 1.4 and 20 ps via a conical intersection, resulting in a 13-cis isomerized retinal. Neither of the two-deactivation channels is significantly preferred. The dynamics is slowed down in comparison with native PR at pH 9 and reaction rates are even lower than for native PR at pH 6, where the primary proton acceptor (Asp97) is protonated. Therefore, the ultrafast isomerization is not only controlled by the charge distribution within the retinal binding pocket. This study shows that in addition to direct electrostatics other effects have to be taken into account to explain the catalytic function of Asp97 in PR on the ultrafast isomerization reaction. This may include sterical interactions and/or bound water molecules within the retinal binding pocket.

Primary Reaction Dynamics of Proteorhodopsin Mutant D97N Observed by Femtosecond Infrared and Visible Spectroscopy†

“…Nevertheless, it is necessary to include τ 1 in the fitting procedure to describe the data appropriately. The subsequent time constants τ 2 (1.6 ± 0.3 ps) and τ 3 (31 ± 3 ps) are similar to the values derived for PR at pD 6.4 in D 2 O (10) and a factor of about 1.3 greater than the time constants achieved for PR D97N under H 2 O conditions (11) (see Table 2). Strong contributions of excited state absorption, ground state bleaching and stimulated emission are found in the respective DAS.…”

“…The UV–Vis spectrum of the solubilized PR D97N in D 2 O shows no deviations from the spectrum measured in H 2 O (11) and is therefore not presented. The absorption maximum of the retinal remains at 550 nm and is shifted by 12 nm compared with PR wt at pD 6.4.…”

“…Expression and purification was performed as described in Lenz et al . (11). The measurements were performed in D 2 O buffer solution (500 m m NaCl and 0.1% n ‐dodecyl‐β‐ d ‐maltoside, 20 m m TRIS at pD 7.4).…”

“…On the one hand, the DAS of τ 1 is not expected to be a mirror image of the photoproduct spectrum, as vibrational relaxation and cooling processes also occur on this timescale. On the other hand, it might only reflect the decay of initially redshifted species, whereas slower reaction pathways as derived from transient absorption measurements in the visible range (11) cannot be verified as these components are obscured by the dominating signature of the decaying bleach signal (10,22).…”

Fluorescence spectroscopy of rhodopsins: Insights and approaches

“…In proteorhodopsin, a mutation (D97N) generated a non-proton pumping variant that shifted the p K a of the SB in a physiological pH range. Weak fluorescence in the near infrared occurred from the protonated SB species while the deprotonated species remained nonfluorescent [36, 37]. This phenomenon – the increase of excited state (fluorescence) lifetime and thus the fluorescence intensity upon protonation of the SB counter ion at low pH or its neutralization by the respective mutant (D85N) – was already observed earlier for bR [31, 38, 39].…”