Decoding the Nitrogenase Mechanism: The Homologue Approach

The (Mo)-nitrogenase is a complex metalloenzyme that catalyzes the key step in the global nitrogen cycle, the reduction of atmospheric dinitrogen (N2) to bioavailable ammonia (NH3), at the iron−molybdenum cofactor (FeMoco) site of its molybdenum−iron (MoFe) protein component. Despite the fundamental significance of biological nitrogen fixation and extensive studies over the past decades, the catalytic mechanism of nitrogenase has not been deciphered. One major challenge for the mechanistic study of nitrogenase is the redox versatility of its FeMoco center. The ability of FeMoco to shuttle between oxidation states in a rapid and unsynchronized manner results in a mixed oxidation state of the cofactor population during turnover. The substrate and the various intermediates can only interact with the FeMoco site in a transient manner, so it is extremely difficult to capture any substrate- or intermediate-bound form of nitrogenase for the direct examination of substrate−enzyme interactions during catalysis. In this Account, we describe the approach of identifying a partially “defective” nitrogenase homologue, one with a slower turnover rate, as a means of overcoming this problem. The NifEN protein complex serves as an ideal candidate for this purpose. It is an α2β2-heterotetramer that contains cluster-binding sites homologous to those found in the MoFe protein: the “P-cluster site” at the interface of the αβ-subunit dimer, which accommodates a [Fe4S4]-type cluster; and the “FeMoco site” within the α-subunit, which houses an all-iron homologue to the FeMoco. Moreover, NifEN mimics the MoFe protein in catalysis: it is capable of reducing acetylene (C2H2) and azide (N3 −) in an ATP- and iron (Fe) protein-dependent manner. However, NifEN is unable to reduce proton (H+) and N2, and it is an inefficient enzyme with a restricted electron flux during the turnover. The extremely slow turnover rate of NifEN and the possible “synchronization” of its FeMoco homologue at a certain oxidation level permit the observation of a new S = 1/2 EPR signal upon turnover of C2H2 by NifEN, which is analogous to the signal reported for a MoFe protein variant upon turnover of the same substrate. This result is exciting, because it suggests the possibility of naturally enriching a C2H2-bound form of NifEN for the successful crystallization of the first intermediate-bound nitrogenase homologue. On the other hand, the fact that NifEN represents a partially “defective” homologue of the MoFe