Nitrogenase reaction mechanism

Non-enzvmatic simulation of nitrogenase reactions and the mechanism of biological nitrogen fixation. G. N. Schrauzer, Angew. Chem., Int. Ed. Engl., 1975, 14, 514-522 (36). 

At present, much attention is devoted to enzymes that utilize the energy of ATP hydrolysis for realization of energy-rich mechanics (myosin), transport (Na+,K+-ATPase, Ca2+-ATPase, chemical processes (nitrogenase), polymerases, topoisomerases, GTPases, and for creation of electrochemical gradients in biomembranes (H+-ATPase, ATP synthase ). In this section we focus on the latter process. The coupling mechanism in the nitrogenase reaction is discussed in Section 3.1. 

In 1970 Likhtenshtein and Shilov advanced the supposition that the enzyme nitrogenase by-passed the above mentioned energy difficulties by realizing a reaction mechanism that provides the rupture of two bonds in N2 with simultaneous compensation due to the formation of four new bonds with catalytic transition atoms. This supposition was based on the following thermodynamic grounds and kinetics considerations. 

Syrtsova, L. A., Druzhinin, S.Y., Rubtsova, E.T., Shkondina, N.I. (1998), New possibilities for studying mechanism of nitrogenase reaction with photodonors of electron, Curr. Plant Sci. Biotechnol Agric. 31 (Biological Nitrogen Fixation for the 21st Century), 49-50. 

Syrtsova, L.A., Druzhinin, S. Yu., Khramov, A. V., and Moravsky, A.P. (1995) Photostimulation of nitrogenase reaction in vitro for investigation of nitrogenase mechanism action Curr. Plant Sci. Biotechnol. Agric. 27 (Nitrogen Fixation Fundamentals and Applicaaaation. 

Fig, 1. Schematic reaction mechanism for substrate reduction by nitrogenase... 

R. A. Alberty, Thermodynamics of the mechanism of the nitrogenase reaction. Biophysical Chemistry, 114, 115-120... 

A starting point for the nitrogenase reaction pathway can be proposed from the mechanisms of N2 reduction catalysed by organometallic complexes. A series of model studies initiated in the early 1960s by the groups of Chatt... 

FIGURE 17.14 Possible reaction mechanisms for nitrogenase. Shown are two possible reaction mechanisms for nitrogenase. On the left is shown the distal mechanism and on the right the alternating mechanism. FeMo-cofactor is abbreviated as M and the names of different bound states are shown. Possible points of entry for diazene and hydrazine are shown. (Adapted fmm Seefeldt et al, 2009.)... 

Nitrogenase Three-Dimensional Structure and Reaction Mechanism 81... 

Figure 12.2.1. Simplified structure of the Fe7Mo-sulfur cluster, found in the MoFe-protein of nitrogenase. The structure was solved by J. Kim and D. C. Rees. Reproduced by permission of the American Association for the Advancement of Science from Science, 1992,257,1677, for the use in Chemical Kinetics and Inorganic Reaction Mechanisms.

A comprehensive description of the mechanism of molybdenum nitrogenase has been provided by the Lowe-Thorneley scheme 102) (Figs. 8 and 9). In this scheme the Fe protein (with MgATP) functions as a single electron donor to the MoFe protein in the Fe protein cycle (Fig. 8), which is broken down into four discrete steps, each of which may be a composite of several reactions ... 

The dependence of rate constants for approach to equilibrium for reaction of the mixed oxide-sulfide complex [Mo3((i3-S)((i-0)3(H20)9] 1+ with thiocyanate has been analyzed into formation and aquation contributions. These reactions involve positions trans to p-oxo groups, mechanisms are dissociative (391). Kinetic and thermodynamic studies on reaction of [Mo3MS4(H20)io]4+ (M = Ni, Pd) with CO have yielded rate constants for reaction with CO. These were put into context with substitution by halide and thiocyanate for the nickel-containing cluster (392). A review of the chemistry of [Mo3S4(H20)9]4+ and related clusters contains some information on substitution in mixed metal derivatives [Mo3MS4(H20)re]4+ (M = Cr, Fe, Ni, Cu, Pd) (393). There are a few asides of mechanistic relevance in a review of synthetic Mo-Fe-S clusters and their relevance to nitrogenase (394). 

Recently we have attempted to pursue multi-electron fixation processes as models for N2 or CO2 fixation. In nature, the N2-fixation enzyme, nitrogenase, exhibits non-specificity properties, and acetylene competes for nitrogen as the fixation substrate (21). The fixation process of acetylene to methane and of nitrogen to ammonia (euqations 14 and 15) have several common features (i) both involve the cleavage of a triple bond (ii) the two reactions involve 6 electrons in the fixation mechanism. Thus, it seems that the photocleavage of acetylene to methane might offer a good model for development of -fixation cycles (22). 

The rate-limiting step of nitrogenase is the dissociation of the reduced MoFe protein from the oxidized Fe protein and has a rate constant k = 6.4 0.8 sec-1. An implicit assumption in the multistep mechanism shown in Figure 13 is that the three elementary reactions coupling each state of the MoFe protein are unperturbed by the reduction level of the large protein. This assumption has been verified for the species E0 and ) [73],... 

This mechanism illustrates another putative role for the hydron in nitroge-nase—as a catalyst for product release. It further illustrates the restrictions in defining a mechanism for substrate conversion by investigating only the enzyme reaction. The involvement of hydrons in the reactions of the nitrogenases can only be inferred from the consumption of electrons, and key catalytic roles for hydrons will not be evident. 

So far the discussion on synthetic clusters illustrates the accumulating information that a variety of nitrogenase substrates can be transformed by simple hydronation reactions at reduced synthetic Fe-S-based clusters. The next level of detail must address the mechanisms of these transformations. Already we have indicated several cases where kinetic studies have been performed. The major problem with the approaches taken so far, looking directly at substrate transformation, is that they can lead to erroneous conclusions. This is because in this approach the experimenter relies on the kinetics to define the number of species essential to accomplish the transformation. For example, the order with respect to hydrons has been established in several of the catalyic systems discussed and invariably found to be one. It is tempting to jump to the conclusion that only one hydron is necessary to activate the cluster. However, studies on the hydronation of Fe-S clusters show that the kinetics of simple hydronation reactions is much more complicated. 

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