Of nitrogenase

Metal clusters in biology quest for a synthetic representation of the catalytic site of nitrogenase. R. H. Holm, Chem. Soc. Rev., 1981,10, 455-490 (137). 

Fe—M—S complexes derived from MS4 anions (M=Mo, W) and their possible relevance as analogues for structural features in the Mo site of nitrogenase. D. Coucouvanis, Acc. Chem. Res., 1981,... 

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). 

The chemistry of nitrogen fixation and models of the reactions of nitrogenase. R. A. Henderson,... 

Not surprisingly, only about 20 of the chemical elements found on Earth are used by living organisms (Chapters 3 and 8). Most of them are common elements. Rare elements are used, if at all, only at extremely low concentrations for specialized functions. An example of the latter is the use of molybdenum as an essential component of nitrogenase, the enzyme that catalyzes the fixation of elemental dinitrogen. Because they are composed of common elements, living organisms exert their most profound effects on the cycles of those elements. 

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. 

In general there are few reproducible data on binding of reducible substrates to the isolated MoFe proteins. However, the S = EPR signal from the FeMoco centers of Kpl is pH dependent, the g values changing with a pKa of 8.7 (50). Of course, the proton is a substrate of nitrogenase however, there is no direct evidence for the proton associated with the pKa being bound directly to FeMoco. Nevertheless, this pKa can be perturbed by addition of the analog substrate acety-... 

Early data on the substrate and inhibitor reactions of nitrogenase were interpreted in terms of five binding sites, with competitive, noncompetitive, unclassified, and negative inhibition being observed (127). This apparent complexity can be readily rationalized in terms of the Lowe—Thorneley scheme (Fig. 9) by assuming that different substrates bind at different oxidation states of the same site. 

The pH dependence of nitrogenase activity has been interpreted in terms of a group with a pi a = 6.3 that must he deprotonated for activity and another group with a pi a = 9 that must be protonated for activity 128). The pi a of the latter group was moved about 0.5 pH units more acid in the presence of acetylene and carbon monoxide and the group with the pi of 6.3 was moved about 0.4 pH units more acid by acetylene. The behavior of the group with the pZa of 9 is fully consistent with earlier observations (50) on the effect of acetylene on... 

Certainly, all three of the bands observed with SF-FTIR must arise from different species, since they appear and disappear with different time courses. The peak at 1904 cm probably corresponds with that observed by ENDOR under low CO conditions, but the relationship of the other two bands to those observed under high CO is not clear, since the ENDOR technique will only detect CO molecules bound to paramagnetic species, whereas FTIR should detect all species. The SF-FTIR technique has the potential to observe the binding and reduction of a wide range of nitrogenase substrates, provided that the appropriate spectroscopic range can be accessed. This will be technically difficult, but well worth the effort. 

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. 

A great deal has been learned about the biosynthesis of nitrogenases, but at the moment the process is understood only in broad outline. The detailed roles of the individual gene products require much further investigation, which may once more indicate fresh approaches to some of the problems identified herein. In particular, if the biosynthetic steps can be emulated chemically, then it may be possible to synthesize FeMoco in large quantities in order to allow its detailed analysis at the atomic level. 

The first active preparations of nitrogenase were isolated in 1966. 

Structure and Function of Nitrogenase Douglas C. Rees, Michael K. Chan, and Jongsun Kim... 

Researchers are working to understand how this enzyme works. Research on nitrogenase takes two main forms. One is an examination of the structure and operation of the enzyme to determine the details of the reactions by which N2 is converted to ammonia. The other form is the s Tithesis of artificial catalysts that mimic the operation of nitrogenase. 

Part 1 of the nitrogenase protein contains another interconnected group of Fe-S atoms, this one with eight iron atoms and seven sulfur atoms. This [8Fe-7S] group collects electrons and transmits them to the binding center. Part 2 of nitrogenase contains a third Fe-S group, this one made up of four iron atoms and four sulfur atoms. This part of the enzyme also binds two molecules of ATP. 

NIS measurements have been performed on the rubredoxin (FeSa) type mutant Rm 2-A from Pyrococcus abyssi [103], on Pyrococcus furiosus rubredoxin [104], on Fe2S2 - and Fe4S4 - proteins and model compounds [105, 106], and on the P-cluster and FeMo-cofactor of nitrogenase [105, 107]. 

Figure 2.10 Schematic structures of (a) sulfite reductase of Escherichia coli in which a 4Fe-4S cluster is linked via a cysteine to the iron in a sirohaem (b) P cluster of nitrogenase (c) FeMoCo cluster of nitrogenase (d) the binuclear site in Desulforibrio gigas hydrogenase.

Hagen, W.R., Eady, R.R., Dunham, W.R., and Haaker, H. 1985b. A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase. FEBS Letters 189 250-254. 

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