The Birch reduction is a widely used synthetic tool to reduce arenes to 1,4-cyclohexadienes. Its harsh cryogenic reaction conditions and the dependence on alkali metals have motivated researchers to explore alternative approaches. In anaerobic aromatic compound degrading microbes, class II benzoyl-coenzyme A (CoA) reductases (BCRs) reduce benzoyl-CoA to the conjugated cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at a tungsten-bis-metallopterin (MPT) cofactor. Though previous structure-based computational studies were in favor of a Birch-like reduction via W(V)/radical intermediates, any experimental evidence for such a mechanism was lacking. Here, we combined freeze-quench and equilibrium electron paramagnetic resonance (EPR) spectroscopic analyses in H2O, D2O, and H217O with redox titrations using wild-type and molecular variants of the catalytic BamB subunit of class II BCR from the anaerobic bacterium Geobacter metallireducens. We provide spectroscopic evidence for a kinetically competent radical/W(V)-OH intermediate obtained after hydrogen atom transfer from the W-aqua-ligand to the aromatic ring and for an invariant histidine as a proton donor assisting the second electron transfer. Quantum mechanical/molecular mechanical calculations suggest that the unique tetrahydro state of both pyranopterins is essential for the reversibility of enzymatic Birch reduction. This work elucidates nature's solution for the chemically demanding Birch reduction and demonstrates how the reactivity of MPT cofactors can be expanded to highly challenging radical chemistry at the negative limit of the biological redox window.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.