Transient Near-UV Absorption of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2: A Spectroscopic Marker for Retinal Configuration

Abstract

We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S 0 → S 3 and/or an S 0 → S 5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2

“…After the extracellular release of the sodium ion, the protein converts back to the initial ground state. Based on mutation studies, spectroscopy and structural data, a number of residues besides the retinal Schiff base (RSB), N112, D116 and Q123 have been identified to be essential for ion uptake and release (1,6,11,12,(14)(15)(16)(17). The S64-Q123 pair seems to form an important gate blocking the Na + entry from the ground state into the ion pathway, which runs along helices B and C. The charged cluster E11-E160-R243 at the extracellular side is either part of the release pathway and/or important for protein stability (1,5,6,11).…”

Photochemistry of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2 and Its Implications on Microbial Rhodopsin Research: Retrospective and Perspective

“…This second bright state (SBS) of 13-cis retinal occurs with the formation of the hot J-intermediate and persists up to the decay of the M-intermediate, suggesting reisomerization occurring during the transition from M to O. 29 A slightly different view is given by recent photocycle studies on NaR and KR2, which show an equilibrium of two O-substates (O 1 and O 2 ) containing either 13-cis (O 1 ) or all-trans (O 2 ) retinal. 32,78,79 Here, the transition from O 1 to O 2 , and hence the isomerization step, is considered to be the molecular switch which leads to vectoriality of the sodium translocation.…”

“…Ultrafast transient absorption experiments on a broad spectrum of microbial rhodopsins have shown that the excited state decay of retinal can often be described by a fast lifetime in the hundreds of fs range and by slower lifetimes which occur on the ps range. ,− The fast lifetime is usually assigned to the transition from the ES to the isomerized GS photoproduct via a conical intersection, whereas the nonreactive pathway is characterized by the slow lifetime components. As a result, two opposing models (and their variants) for the primary dynamics are mainly discussed in the broad community: The ES branching (Figure a) and the GS heterogeneity (Figure b) model. ,,,− The surprisingly fast lifetime of around 200 fs in the case of KR2, − combined with its structural uniqueness in the vicinity of the chromophore, revived the interest in this debate. Without a detailed look at those models and their nuances, which would be out of scope of this article, we explicitly focus on a few key aspects.…”

“…Its overall similarity to other microbial rhodopsins raised questions toward structural and mechanistic nuances, which allow the active transport of a positive charge against a large electrostatic barrier inside the protein . This has been intensely investigated with various time-resolved techniques which cover the protein dynamics from the primary reaction of photon absorption, − via the energy transfer from the retinal to the protein backbone , to the uptake, translocation and release of the sodium ion. ,, In this article we will focus on the main advances in our understanding of the KR2 mechanism from a spectroscopic point of view.…”

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

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