Nitrogenase enzymes structure

In this chapter we hrst review the chemistry of the Fe-S sites that occur in relatively simple rubredoxins and ferredoxins, and make note of the ubiquity of these sites in other metalloenzymes. We use these relatively simple systems to show the usefulness of spectroscopy and model-system studies for deducing bioinorganic structure and reactivity. We then direct our attention to the hydrogenase and nitrogenase enzyme systems, both of which use transition-metal-sulfur clusters to activate and evolve molecular hydrogen. 

The iron-sulfur proteins, the ferredoxins, are so ubiquitous that it is difficult to realize that they were discovered only in the 1960s because of their unusual (at that time) g values. The simplest, rubredoxin, contains only one iron with the sulfur atoms of four cysteines supplying the ligation. Numerous others contain pairs of Fe atoms, and yet others contain 4Fe-4S cubes. The magnetic couplings between iron atoms leads to the unusual EPR g values, and a review by their discoverer is worthy of perusal. Iron and molybdenum XAS as well as EPR provided important clues to the structure of the cores of the nitrogenase enzyme prior to its X-ray crystal structure determination, as described in [26]. 

In 1930, Hermann Bortels (1902-1979) recognised that nitrogen fixation is a molybdenum-dependent process. Obviously, the nitrogenases from Rhizobium meliloti, Azotobacter vinelandii and Clostridium pasteurianum have a similar constitution. In 1966, Leonard E. Mortenson identified for the first time an Fe- and a MoFe-protein as parts of the nitrogenase enzyme system. The exact structure of the nitrogenase-molybdenum-iron protein from Azotobacter vinelandii [28] was clarified in 1992, and that from Clostridium pasteurianum [29] in 1993, both by Douglas C. Rees. [30] The Fe-protein is a y2-dimer with a molar mass of some 60,000 Daltons, and the MoFe-protein is an ca. 

HIV-protease (Fig. 21.11) is the target of one kind of AIDS drugs, as discussed in Chap. 17. Figure 21.12 is nitrogenase, one of the most complex enzymes, and is talked about in Chap. 6. Determination of protein (enzyme) structures as illustrated here and others help enormously in understanding their functions and the mechanisms in which they play crucial roles. 

In our initial publications on this topic we demonstrated that FeMoco exchanges electrons with an electrode and that its electrochemical behavior is qualitatively consistent with the previously determined redox behavior of the Mo-Fe-S center in intact nitrogenase enzyme . In this paper we present the results of a more detailed electrochemical examination of FeMoco and provide evidence for the existence of multiple forms of cofactor in its fully oxidized and semi-reduced oxidation states and for electrocatalytic activity associated with reduction to its fully reduced level. Both observations are significant in that they may lead to greater understanding of the structure and function of FeMoco. 

These were relatively low-resolution structures, and with refinement some errors in the initial structural assignments have been detected (4-7). Since the structures were first reported the subject has been extensively reviewed in this series (8) and elsewhere 9-15). This review will focus on the structure, biosynthesis, and function of the met-allosulfur clusters found in nitrogenases. This will require a broader overview of some functional aspects, particularly the involvement of MgATP in the enzymic reaction, and also some reference will be made to the extensive literature (9, 15) on biomimetic chemistry that has helped to illuminate possible modes of nitrogenase function, although a detailed review of this chemistry will not be attempted here. This review cannot be fully comprehensive in the space available, but concentrates on recent advances and attempts to describe the current level of our understanding. 

A preparation of the third nitrogenase from A. vinelandii, isolated from a molybdenum-tolerant strain but lacking the structural genes for the molybdenum and vanadium nitrogenases, was discovered to contain FeMoco 194). The 8 subunit encoded by anfG was identified in this preparation, which contained 24 Fe atoms and 1 Mo atom per mol. EPR spectroscopy and extraction of the cofactor identified it as FeMoco. The hybrid enzyme could reduce N2 to ammonia and reduced acetylene to ethylene and ethane. The rate of formation of ethane was nonlinear and the ethane ethylene ratio was strongly dependent on the ratio of nitrogenase components. 

The elucidation of the crystal structures of two high-spin EPR proteins has shown that the proposals for novel Fe-S clusters are not without substance. Two, rather than one novel Fe-S cluster, were shown to be present in nitrogenase, the key enzyme in the biotic fixation of molecular nitrogen 4, 5). Thus the FeMoco-cofactor comprises two metal clusters of composition [4Fe-3S] and [lMo-3Fe-3S] bridged by three inorganic sulfur atoms, and this is some 14 A distant from the P-cluster, which is essentially two [4Fe-4S] cubane moieties sharing a corner. The elucidation of the crystal structure of the Fepr protein (6) provides the second example of a high-spin EPR protein that contains yet another unprecedented Fe-S cluster. 

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