Seven Steps
of Alternating Electron and Proton Transfer
in Photosystem II Water Oxidation Traced by Time-Resolved Photothermal
Beam Deflection at Improved Sensitivity
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Abstract
The
intricate orchestration of electron transfer (ET) and proton
transfer (PT) at the Mn<sub>4</sub>CaO<sub><i>n</i></sub>-cluster of photosystem II (PSII) is mechanistically pivotal but
clearly insufficiently understood. Preparations of PSII membrane particles
were investigated using a kinetically competent and sensitive method,
photothermal beam deflection (PBD), to monitor apparent volume changes
of the PSII protein. Driven by nanosecond laser flashes, the PSII
was synchronously stepped through its water-oxidation cycle involving
four (semi)stable states (S<sub>0,</sub> S<sub>1</sub>, S<sub>2</sub>, and S<sub>3</sub>) and minimally three additional transiently formed
intermediates. The PBD approach was optimized as compared to our previous
experiments, resulting in superior signal quality and resolution of
more reaction steps. Now seven transitions were detected and attributed,
according to the H/D-exchange, temperature, and pH effects on their
time constants, to ET or PT events. The ET steps oxidizing the Mn<sub>4</sub>CaO<sub><i>n</i></sub> cluster in the S<sub>2</sub> → S<sub>3</sub> and S<sub>0</sub> → S<sub>1</sub> transitions,
a biphasic PT prior to the O<sub>2</sub>-evolving reaction, as well
as the reoxidation of the primary quinone acceptor (Q<sub>A</sub><sup>–</sup>) at the PSII acceptor side were detected for the first
time by PBD. The associated volume changes involve (i) initial formation
of charged groups resulting in contraction assignable to electrostriction,
(ii) volume contraction explainable by reduced metal–ligand
distances upon manganese oxidation, and (iii) charge-compensating
proton removal resulting in volume expansion due to electrostriction
reversal. These results support a reaction cycle of water oxidation
exhibiting alternate ET and PT steps. An extended kinetic scheme for
the O<sub>2</sub>-evolving S<sub>3</sub> ⇒ S<sub>0</sub> transition
is proposed, which includes crucial structural and protonic events