3/2. E3 – This intermediate is proposed to be the singly reduced FeMo-co with one bridging hydride and one proton. E4 – Termed the Janus intermediate after the Roman god of transitions, this intermediate is positioned after exactly half of the electron proton transfers and can either decay back to E0 or proceed with nitrogen binding and finish the catalytic cycle. This intermediate is proposed to contain the FeMo-co in its resting oxidation state with two bridging hydrides and two sulfur bonded protons. This intermediate was first observed using freeze quench techniques with a mutated protein in which residue 70, a valine amino acid, is replaced with isoleucine. This modification prevents substrate access to the FeMo-co. EPR characterization of this isolated intermediate shows a new species with a spin of ½. ENDOR experiments have provided insight into the structure of this intermediate, revealing the presence of two bridging hydrides. 95Mo and 57Fe ENDOR show that the hydrides bridge between two iron centers. Cryoannealing of the trapped intermediate at -20 °C results in the successive loss of two hydrogen equivalents upon relaxation, proving that the isolated intermediate is consistent with the E4 state. The decay of E4 to E2 + H2 and finally to E0 and 2H2 has confirmed the EPR signal associated with the E2 intermediate. The above intermediates suggest that the metal cluster is cycled between its original oxidation state and a singly reduced state with additional electrons being stored in hydrides. It has alternatively been proposed that each step involves the formation of a hydride and that the metal cluster actually cycles between the original oxidation state and a singly oxidized state. Distal and alternating pathways for N2 fixation While the mechanism for nitrogen fixation prior to the Janus E4 complex is generally agreed upon, there are currently two hypotheses for the exact pathway in the second half of the mechanism: the "distal" and the "alternating" pathway (see Figure 5). In the distal pathway, the terminal nitrogen is hydrogenated first, releases ammonia, then the nitrogen directly bound to the metal is hydrogenated. In the alternating pathway, one hydrogen is added to the terminal nitrogen, then one hydrogen is added to the nitrogen directly bound to the metal. This alternating pattern continues until ammonia is released. Because each pathway favors a unique set of intermediates, attempts to determine which path is correct have generally focused on the isolation of