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Using models derived from the X-ray structure of photosystem II, it is shown that the oxygen evolving complex in the S(2) state exists in two energetically similar and interconvertible forms. A longstanding question regarding the spectroscopy of the catalyst is thus answered: one form corresponds to the multiline g=2.0 EPR signal (see picture, right; O red, Mn purple, Ca yellow), and the other to the g≥4.1 signals (left).

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake on earth 1,2 and are thus extensively studied with respect to their technological exploitation as noble metal substitutes in (photo)electrolysers and fuel cells [3][4][5] . In [FeFe]-hydrogenases catalysis takes place at a unique diiron center (the [2Fe] subsite) featuring a bridging dithiolate ligand, as well as three CO and two CN − ligands (Figure 1) 6,7 . Through a complex and as yet poorly understood multienzymatic biosynthetic process, this [2Fe] subsite is first assembled onto a maturation enzyme, HydF. From there, it is delivered to the apo-hydrogenase for activation 8 . Synthetic chemistry has allowed the preparation of remarkably close mimics of that subsite 1 but failed to reproduce the natural enzymatic activities so far. Here we show that three such synthetic mimics (with different bridging dithiolate ligands) can be loaded onto HydF and then transferred to apoHydA1, one of the hydrogenases of Chlamydomonas reinhardtii. Remarkably, full activation of HydA1 was achieved exclusively using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of 10 . This is the first example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe] cluster 17 and named "HydF" in the following, with a 10-fold molar excess of complex 1, 2 or 3, led to new hybrid species x-HydF (x = 1, 2 or 3 respectively), that could be isolated in pure form and characterized. In all cases, iron quantification indeed showed an increase from 3.9 ± 0.4 to 5.6 ± 0.4 iron atoms per protein and the UV-visible spectrum of these hybrids displayed features consistent with a ~1:1 ratio of the synthetic complexes and the HydF protein ( Figure S1a-c).FTIR spectroscopy is a convenient method for characterizing metalloproteins such as hydrogenases containing CO and CN − ligands 18 . Thus, further evidence for the incorporation of synthetic complexes in HydF was obtained from their FTIR spectra which contained CN − stretching bands between 2000 and 2100 cm −1 and four partly overlapping CO-stretching bands in the 1800-2000 cm −1 range ( Figure 2B and Table S1). The highenergy bands underwent a 40 cm −1 shift upon 13 C-labeling of the CN − ligands ( Figure S2). Interestingly, the width of the FTIR bands is still identical to those of the unbound complexes ( Figure 2A) but their positions show strong similarities with those of CaHydF ( Figure 2B and The arrangement in which the synthetic complexes are bound to HydF and its [4Fe-4S] cluster is not evident from the FTIR spectra. In particular FTIR spectroscopy does not allow to definitively distinguish between terminal and bridging cyanide ligands (see below and supplementary discussion) ...

The photosynthetic protein complex photosystem II oxidizes water to molecular oxygen at an embedded tetramanganese-calcium cluster. Resolving the geometric and electronic structure of this cluster in its highest metastable catalytic state (designated S3) is a prerequisite for understanding the mechanism of O-O bond formation. Here, multifrequency, multidimensional magnetic resonance spectroscopy reveals that all four manganese ions of the catalyst are structurally and electronically similar immediately before the final oxygen evolution step; they all exhibit a 4+ formal oxidation state and octahedral local geometry. Only one structural model derived from quantum chemical modeling is consistent with all magnetic resonance data; its formation requires the binding of an additional water molecule. O-O bond formation would then proceed by the coupling of two proximal manganese-bound oxygens in the transition state of the cofactor.

Photosystem II (PSII), a multisubunit pigment-protein supercomplex found in cyanobacteria, algae, and plants, catalyzes a unique reaction in nature: the light-driven oxidation of water. Remarkable recent advances in the structural analysis of PSII now give a detailed picture of the static supercomplex on the molecular level. These data provide a solid foundation for future functional studies, in particular the mechanism of water oxidation and oxygen release. The catalytic core of the PSII is a tetramanganese-calcium cluster (Mn₄O₅Ca), commonly referred to as the oxygen-evolving complex (OEC). The function of the OEC rests on its ability to cycle through five metastable states (Si, i = 0-4), transiently storing four oxidizing equivalents, and in so doing, facilitates the four electron water splitting reaction. While the latest crystallographic model of PSII gives an atomic picture of the OEC, the exact connectivity within the inorganic core and the S-state(s) that the X-ray model represents remain uncertain. In this Account, we describe our joint experimental and theoretical efforts to eliminate these ambiguities by combining the X-ray data with spectroscopic constraints and introducing computational modeling. We are developing quantum chemical methods to predict electron paramagnetic resonance (EPR) parameters for transition metal clusters, especially focusing on spin-projection approaches combined with density functional theory (DFT) calculations. We aim to resolve the geometric and electronic structures of all S-states, correlating their structural features with spectroscopic observations to elucidate reactivity. The sequence of manganese oxidations and concomitant charge compensation events via proton transfer allow us to rationalize the multielectron S-state cycle. EPR spectroscopy combined with theoretical calculations provides a unique window into the tetramangenese complex, in particular its protonation states and metal ligand sphere evolution, far beyond the scope of static techniques such as X-ray crystallography. This approach has led, for example, to a detailed understanding of the EPR signals in the S₂-state of the OEC in terms of two interconvertible, isoenergetic structures. These two structures differ in their valence distribution and spin multiplicity, which has important consequences for substrate binding and may explain its low barrier exchange with solvent water. New experimental techniques and innovative sample preparations are beginning to unravel the complex sequence of substrate uptake/inclusion, which is coupled to proton release. The introduction of specific site perturbations, such as replacing Ca²⁺ with Sr²⁺, provides discrete information about the ligand environment of the individual Mn ions. In this way, we have identified a potential open coordination site for one Mn center, which may serve as a substrate binding site in the higher S-states, such as S₃ and S₄. In addition, we can now monitor the binding of the substrate water in the lower S-states (S₁ and S₂) using new EPR-detected N...