Decoding the Nitrogenase Mechanism: The Homologue Approach

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• 作者：Yilin Hu ; Markus W. Ribbe
• 刊名：Accounts of Chemical Research
• 出版年：2010
• 出版时间：March 16, 2010
• 年：2010
• 卷：43
• 期：3
• 页码：475-484
• 全文大小：512K
• 年卷期：v.43,no.3(March 16, 2010)
• ISSN：1520-4898

文摘

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 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 N₂ 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.