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 (N(2)) to bioavailable ammonia (NH(3)), 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 alpha(2)beta(2)-heterotetramer that contains cluster-binding sites homologous to those found in the MoFe protein: the "P-cluster site" at the interface of the alphabeta-subunit dimer, which accommodates a [Fe(4)S(4)]-type cluster; and the "FeMoco site" within the alpha-subunit, which houses an all-iron homologue to the FeMoco. Moreover, NifEN mimics the MoFe protein in catalysis: it is capable of reducing acetylene (C(2)H(2)) and azide (N(3)(-)) in an ATP- and iron (Fe) protein-dependent manner. However, NifEN is unable to reduce proton (H(+)) and N(2), 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 C(2)H(2) 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 C(2)H(2)-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 protein makes it a promising mutational platform on which a functional MoFe protein equivalent may be reconstructed by introducing the missing features of MoFe protein step-by-step into NifEN. Such a strategy allows us to define the function of each feature and address questions such as the following: What is the function of P-cluster in catalysis? Are Mo and homocitrate the essential constituents of the cofactor in N(2) reduction? How does substrate accessibility affect the reactivity of the enzyme? This homologue approach could complement the mechanistic analysis of the nitrogenase MoFe protein, and information derived from both approaches will help achieve the ultimate goal of solving the riddle of biological nitrogen fixation.
Hu
Yilin
Y
Department of Molecular Biology and Biochemistry, University of California-Irvine, 2236 McGaugh Hall, Irvine, California 92697-3900, USA. yilinh@uci.edu
Ribbe
Markus W
MW
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation
D008983:Molybdoferredoxin; D009591:Nitrogenase