Nature’s water splitting cofactor passes through a series of catalytic intermediates (S₀-S₄) before O-O bond formation and O₂ release. In the second last transition (S₂ to S₃) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all Mn^IV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S₄. How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S₃9) in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (>50%) in (i) chemical-modified cofactors in which Ca²⁺ is exchanged with Sr²⁺; the Mn₄O₅Sr cofactor remains active, but the S₂-S₃ and S₃-S₀ transitions are slower than for the Mn₄O₅Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S₃9 electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S₃ signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S₂ state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S₃9 cofactor is similar to the high spin form of the preceding S₂ state, which contains a cuboidal Mn₃O₄Ca unit tethered to an external, 5-coordinate Mn ion (Mn4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn4. The interaction of ammonia with the cofactor is also discussed. Significance Recent results have shown that nature’s water splitting catalyst inserts an additional water molecule into what appears to be a solvent inaccessible site late in its reaction cycle. The emerging consensus of the field is that this water molecule is one of the substrates of the reaction. Here, we show that this water molecule does not come directly from solvent. It instead represents an earlier bound water, which is inserted into this site via facile structural tautomerism. The trigger for this process is cofactor oxidation. This then allows an additional water to bind from solvent to a more open site of the cofactor. In this way the cofactor carefully regulates water uptake, preventing water insertion earlier in the reaction cycle.

Here we report an in situ electron paramagnetic resonance (EPR) study of a low-cost, high-stability cobalt oxide electrodeposited material (Co-Pi) that oxidizes water at neutral pH and low over-potential, representing a promising system for future large-scale water splitting applications. Using CW X-band EPR we can follow the film formation from a Co(NO₃)₂ solution in phosphate buffer and quantify Co uptake into the catalytic film. As deposited, the film shows predominantly a Co(II) EPR signal, which converts into a Co(IV) signal as the electrode potential is increased. A purpose-built spectroelectrochemical cell allowed us to quantify the extent of Co(II) to Co(IV) conversion as a function of potential bias under operating conditions. Consistent with its role as an intermediate, Co(IV) is formed at potentials commensurate with electrocatalytic O₂ evolution (+1.2 V, vs. SHE). The EPR resonance position of the Co(IV) species shifts to higher fields as the potential is increased above 1.2 V. Such a shift of the Co(IV) signal may be assigned to changes in the local Co structure, displaying a more distorted ligand field or more ligand radical character, suggesting it is this subset of sites that represents the catalytically ‘active’ component. The described spectroelectrochemical approach provides new information on catalyst function and reaction pathways of water oxidation.

Singlet vinylidenes (R₂C═C:) are proposed as intermediates in a series of organic reactions, and very few have been studied by matrix isolation or gas-phase spectroscopy. Triplet vinylidenes, however, featuring two unpaired electrons at a monosubstituted carbon atom are thus far only predicted as electronically excited-state species and represent an unexplored class of carbon-centered diradicals. We report the photochemical generation and low-temperature EPR/ENDOR characterization of the first ground-state high-spin (triplet) vinylidene. The zero-field splitting parameters (D = 0.377 cm^–1 and |E|/D = 0.028) were determined, and the ¹³C hyperfine coupling tensor was obtained by ¹³C-ENDOR measurements. Most strikingly, the isotropic ¹³C hyperfine coupling constant (50 MHz) is far smaller than the characteristic values of triplet carbenes, demonstrating a unique electronic structure which is supported by quantum chemical calculations.

The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y₁₂₂^.) cofactor for activity. The knowledge on the in vitro Y₁₂₂^. structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y₁₂₂^.. Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y₁₂₂^. are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y₁₂₂^. per β2 distribution, supporting the key role of Y^. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F₃Y₁₂₂^., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.