Life science
Light energy conversion

Elucidation of the pumping mechanismof light-driven proton-pumping proteins
Department of Chemistry, Graduate School of Science
Professor Yasuhisa Mizutani
https://researchmap.jp/read0182663?lang=en

Life science

Abstract
In living organisms, energy conversion and material transport
occur via proton concentration gradients across membranes.
These gradients are established by proteins that actively transport
protons, known as proton pumps. Some microorganisms havebeen found to use outward-facing proton pumps that utilize lightenergy to transport protons from the cell interior to the exterior.
Recently, a protein that inwardly transports protons, in opposition
to conventional proteins, has been discovered and garnered considerable attention, despite sharing a similar three-dimensional
structure. Nevertheless, the mechanism of inward proton transportremains unclear. We observed the structural changes of the retinal
chromophore in schizorhodopsin 4, an inward proton pump, anddetermined that the sequence of the structural changes is reversed
compared to that of conventional proteins, which allows for thetransportation of protons in the opposite direction.

▍ Background & Results
We employed time-resolved resonance Raman spectroscopy toobserve the structural changes in the retinal chromophore of schizorhodopsin 4. The conventional proton pumps undergo isomerization upon light absorption, deprotonation of the Schiff base, followed by reprotonation, and finally reisomerization. This is shown in
Figure 2 (left). However, our study on schizorhodopsin 4 indicatesthat reisomerization and subsequent reprotonation take place after
the deprotonation of the Schiff base (Figure 2, right). Most notably,
the sequence of reisomerization and reprotonation is reversed.
Additionally, this difference can explain why schizorhodopsin 4
pumps protons in the opposite direction to conventional proton
pumps. Conventional proton pumps release protons following photoisomerization of Schiff bases towards the cytoplasmic medium.
However, transportation of the protons to the extracellular medium
is necessary, and it is believed that the negatively charged aminoacid residues on the extracellular side of the chromophore attract
the proton released from the Schiff base. The Schiff base that has
lost the proton maintain their orientation and accepts a proton fromthe cytoplasmic medium. Finally, the Schiff base faces extracellular medium upon reisomerization. Schizorhodopsin 4 releases aproton to cytoplasmic medium after the Schiff base is flipped uponisomerization. Then, the chromophore is subsequently reisomerized. The Schiff base is oriented towards the extracellular medium,receiving protons from extracellular medium. Accordingly, the proton transport mechanism has been found to be highly rational forthe inward proton transport.

Figure1▍ Significance of the research and Future perspective
The results of this study indicate that the sequence of structuralchanges of the chromophore has an influence on the directionof proton transport. This finding is expected to facilitate the understanding of the principle of proton transport and promote themolecular design of proton pumps by revealing and analyzing themechanisms of other inward-directed proton pumps.

Figure2

P a t e n t
Hayashi, Kouhei; Mizuno, Misao; Mizutani, Yasuhisa et al. Cis-Trans reisomerization precedes reprotonation of the retinal chromophore in the photocycle ofschizorhodopsin 4, Angew. Chem. Int. Ed. 2022, 61, e202203149. doi: 10.1002/anie.202203149
URL https://www.chem.sci.osaka-u.ac.jp/lab/mizutani/index-e.html
K e y w o r d light energy conversion, proton transfer, protein dynamics, time-resolved spectroscopy
Tr e a t i s e