pH-Dependent Photoisomerization of Retinal in Proteorhodopsin

Huber, Robert; Köhler, Thorsten; Lenz, Martin O.; Bamberg, Ernst; Kalmbach, Rolf; Engelhard, Martin; Wachtveitl, Josef

doi:10.1021/bi048318h

Cited by 75 publications

(173 citation statements)

References 43 publications

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“…Transient Visible-pump/Visible-probe Spectroscopy-The experimental setup for the pump/probe spectroscopy in the visible spectral range was described in detail elsewhere (23). In brief, the pulse source for these experiments was a Clark CPA 2001 femtosecond laser system (central wavelength 775 nm, pulse length 170 fs, pulse energy 800 J, repetition rate1 kHz).…”

Section: Methodsmentioning

confidence: 99%

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Staudt

Oesterhelt

Grininger

et al. 2012

Journal of Biological Chemistry

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Background: Dodecin prevents riboflavin from autodegradation and exerting photo-induced cellular stress. Results: Ultrafast depopulation of excited states and ground state recovery is observed by transient spectroscopy. Conclusion: Ultrafast electron transfer in combination with proton transfer is responsible for deactivation of flavin excited states. Significance: A comprehensive study of parameters in the binding pocket affecting the riboflavin quenching mechanism is given.

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Section: Methodsmentioning

confidence: 99%

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Staudt

Oesterhelt

Grininger

et al. 2012

Journal of Biological Chemistry

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“…Oxygenfree conditions were achieved by several cycles of degassing the reaction solution under argon atmosphere. Femtosecond pump/probe experiments were performed as described elsewhere (17).…”

Section: Preparation Of Vectors and Strains And Purification Ofmentioning

confidence: 99%

Dodecin Is the Key Player in Flavin Homeostasis of Archaea

Grininger

Staudt

Johansson

et al. 2009

Journal of Biological Chemistry

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Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.Flavins are a major class of cofactors that are able to accept and donate electrons as well as to absorb visible light. Flavins consist of a nonconserved aliphatic moiety, covalently linked to a conserved aromatic isoalloxazine ring ( In all flavins, the catalytically active unit is the isoalloxazine substructure (2-4). To handle the reactivity of the isoalloxazine ring, FMN and FAD are tightly bound to flavoenzymes. Finely tuned binding restricts the reaction spectrum of FMN and FAD to discrete, beneficial reaction pathways, preventing harmful side reactions. Reported functions of flavoenzymes include transferring electrons from and to reactive centers (e.g. respiratory chain) as well as employing light for the induction of radical reactions (e.g. DNA-photolyase) or conformational changes (e.g. phototropin) (5-8). In the biosynthesis, the full catalytic power of flavins has already formed at the stage of the FMN and FAD precursor RF (see Fig. 1). As RF is not used as an enzymatically active compound, it is, different to FMN and FAD, not integrated into flavoenzymes. To avoid a negative impact of uncontrolled flavin reactivity by free RF, nature established sequestering devices termed riboflavin-binding proteins (RfBPs). These proteins have been shown to store and distribute RF in eukaryotes, ensuring a fast supply of FMN and FAD (9, 10).The small flavoprotein dodecin from the archaeon Halobacterium salinarum was reported to bind RF with high affinity and to extensiv...

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“…Data detection was implemented in a referenced scheme using two 42-segment photodiode arrays providing a typical resolution of 10 À4 OD units. [57] An instrumental response function of~80 fs (NOPA) and 150 fs (SFM) was achieved and a spectral range from 380 to 710 nm was covered. To ensure the exchange of the probed sample volume between individual excitation pulses, the fused silica cuvette with 1 mm optical pathlength was moved laterally with appropriate speed.…”

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confidence: 99%

The Photocycle of Channelrhodopsin‐2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps

et al. 2010

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The photocycle of channelrhodopsin-2 is investigated in a comprehensive study by ultrafast absorption and fluorescence spectroscopy as well as flash photolysis in the visible spectral range. The ultrafast techniques reveal an excited-state decay mechanism analogous to that of the archaeal bacteriorhodopsin and sensory rhodopsin II from Natronomonas pharaonis. After a fast vibrational relaxation of the excited-state population with 150 fs its decay with mainly 400 fs is observed. Hereby, both the initial all-trans retinal ground state and the 13-cis-retinal K photoproduct are populated. The reaction proceeds with a 2.7 ps component assigned to cooling processes. Small spectral shifts are observed on a 200 ps timescale. They are attributed to conformational rearrangements in the retinal binding pocket. The subsequent dynamics progresses with the formation of an M-like intermediate (7 and 120 μs), which decays into red-shifted states within 3 ms. Ground-state recovery including channel closing and reisomerization of the retinal chromophore occurs in a triexponential manner (6 ms, 33 ms, 3.4 s). To learn more about the energy barriers between the different photocycle intermediates, temperature-dependent flash photolysis measurements are performed between 10 and 30°C. The first five time constants decrease with increasing temperature. The calculated thermodynamic parameters indicate that the closing mechanism is controlled by large negative entropy changes. The last time constant is temperature independent, which demonstrates that the photocycle is most likely completed by a series of individual steps recovering the initial structure.

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pH-Dependent Photoisomerization of Retinal in Proteorhodopsin

Cited by 75 publications

References 43 publications

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Dodecin Is the Key Player in Flavin Homeostasis of Archaea

The Photocycle of Channelrhodopsin‐2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps

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