Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel–production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese–calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S ₂ → S ₃ transition of the classical S -state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant ( ). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein–water interface is characterized by a high activation energy ( E _a = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S ₀ → S ₁ transition are similar (τ, approximately 100 µs; E _a = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

The water-oxidation catalytic activity of [Ru^II(damp)(bpy)(H₂O)]²⁺ has been determined from manometric and mass spectroscopy studies. Mechanistic details of the catalytic cycle have been studied both experimentally and using DFT and CASSCF/CASPT2 calculations. Characterisation of this Ru(II) complex and more highly oxidized catalytic intermediates has been accomplished through UV-vis and XAS spectroscopy, as well as through electrochemical techniques. Comparison of XAS spectra with CASSCF/CASPT2 calculations provides insight into the electronic structures of the more highly oxidized species, especially the degree to which oxidation occurs over both atoms of the Ru–O fragment. ¹⁸O-labelling experiments indicate that the O–O bond formation step proceeds via a water nucleophilic attack mechanism, and a detailed DFT analysis of the catalytic cycle predicts that step to be rate-determining and to take place for a formal Ru(V)O species. A number of alternative higher energy pathways have also been characterised in order to provide a more complete vision of the whole system.

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe–4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN⁻) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN⁻ vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred′ represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred′ was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN⁻ infrared band patterns favored a cysteine ligand of the [4Fe–4S] cluster as the protonation site in HoxH and Hred′. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe–4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe–4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Irreversible inhibition by molecular oxygen (O₂) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H₂) production. Modification by O₂ of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe_H) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O₂ exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O₂ reactions. The first phase (τ₁ ≤ 4 s) is characterized by the formation of an increased number of Fe–O,C bonds, elongation of the Fe–Fe distance in the binuclear unit (2Fe_H), and oxidation of one iron ion. The second phase (τ₂ ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-Å Fe–Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ₃ ≤ 1000 s) leads to the disappearance of most Fe–Fe and Fe–S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe_H⁺ leading to the formation of a reactive oxygen species-like superoxide (O₂⁻), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S]⁺ unit, leading to 3) complete O₂-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O₂ tolerance in [FeFe]-hydrogenase.