Hydrogenases couple electrochemical potential to the reversible chemical transformation of H and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]) with CO and CN ligands. Mössbauer analysis and density functional theory (DFT) calculations show that H consists of a reduced [4Fe-4S] coupled to a diferrous [2Fe] with a terminally bound Fe-hydride. The existence of the Fe-hydride in H was demonstrated by an unusually low Mössbauer isomer shift of the distal Fe of the [2Fe] subcluster. A DFT model of H shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.

[FeIV(O)(TMG3tren)]2+ (1; TMG3tren = 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine) is a unique example of an isolable synthetic S = 2 oxoiron(IV) complex, which serves as a model for the high-valent oxoiron(IV) intermediates observed in nonheme iron enzymes. Congruent with DFT calculations predicting a more reactive S = 2 oxoiron(IV) center, 1 has a lifetime significantly shorter than related S = 1 oxoiron(IV) complexes. Self-decay of 1 exhibits strictly first-order kinetic behavior and is unaffected by solvent deuteration, suggesting an intramolecular process. This hypothesis was supported by ESI-MS analysis of the iron products and a significant retardation of self-decay upon use of a perdeuteromethyl TMG3tren isotopomer, d36-1 (KIE = 24 at 25°C). The greatly enhanced thermal stability of d36-1 allowed growth of diffraction quality crystals for which a high-resolution crystal structure was obtained. This structure showed an Fe=O unit (r = 1.661(2) Å) in the intended trigonal bipyramidal geometry enforced by the sterically bulky tetramethylguanidinyl donors of the tetradentate tripodal TMG3tren ligand. The close proximity of the methyl substituents to the oxoiron unit yielded three symmetrically oriented short C-D···O non-bonded contacts (2.38 – 2.49 Å), an arrangement that facilitated self-decay by rate-determining intramolecular hydrogen atom abstraction and subsequent formation of a ligand-hydroxylated iron(III) product. EPR and Mössbauer quantification of the various iron products, referenced against those obtained from reaction of 1 with 1,4-cyclohexadiene, allowed formulation of a detailed mechanism for the self-decay process. The solution of this first crystal structure of a high-spin (S = 2) oxoiron(IV) center represents a fundamental step on the path towards a full understanding of these pivotal biological intermediates.

The bicyclic β-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of L-proline followed by β-lactam ring closure. Carbapenem synthase (CarC), an Fe(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.