Spin Polarization Reveals the Coordination Geometry of the [FeFe] Hydrogenase Active Site in Its CO-Inhibited State.

Edward Reijerse,Wolfgang Lubitz,James A Birrell

doi:10.1021/acs.jpclett.0c01352

Edward Reijerse, Wolfgang Lubitz + Show 1 more

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https://doi.org/10.1021/acs.jpclett.0c01352

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Abstract

The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe2ADT(CO)3(CN)2, where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN– bound to the distal Fe becomes apical. Using selective 13C isotope labeling of the CN– and COext ligands in combination with pulsed 13C electron–nuclear–nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the “unrotated” apical COext configuration.

Highlights

The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe2ADT(CO)3(CN)[2], where ADT represents the bridging ligand aza-propane-dithiolate
It is generally assumed that the oxidized state of the H-cluster, “Hox”, is the entry point to the catalytic cycle.[2]
To further corroborate the assignment of Hox-CO to conformation “S-COa” with COext in the apical position, we set out to determine the relative signs of the 13C hyperfine interactions (HFIs) tensors and performed electron−nuclear−nuclear triple resonance (TRIPLE)[20] experiments using Chlamydomonas reinhardtii (CrHydA1) samples obtained through artificial maturation with synthetic precursors labeled with 13CN−21,22 and inhibited with either natural abundance CO or 13CO

Summary

The Journal of Physical Chemistry Letters

To further corroborate the assignment of Hox-CO to conformation “S-COa” with COext in the apical position, we set out to determine the relative signs of the 13C HFI tensors and performed electron−nuclear−nuclear triple resonance (TRIPLE)[20] experiments (see the Supporting Information and Scheme S1) using Chlamydomonas reinhardtii (CrHydA1) samples obtained through artificial maturation with synthetic precursors labeled with 13CN−21,22 and inhibited with either natural abundance CO or 13CO (see Figure S1 for FTIR spectra of the samples). From the difference spectrum colored blue, one can see that the “triple effect” occurs on the lowfrequency line of the 13CN− outer doublet as well as on the high-frequency Mims TRIPLE rleinsueltotfhatht eth1e3CtwOoex1t3CligNa−ndli.gaTnhdisHcFoInsfihrmavse the the same sign.

■ ACKNOWLEDGMENTS

■ REFERENCES