Nitrogenase inhibitors

In contrast to the situation with the alternative nitrogenases, but with the notable exception of the C. pasteurianum proteins, the component proteins from aU. Mo-based nitrogenases interact as heterologous crosses to form catalyticaHy active enzymes (52). Carbon monoxide, CO, is a potent inhibitor of aU. nitrogenase-cataly2ed substrate reductions, with the exception of reduction (126). Molecular hydrogen has a unique involvement with Mo-nitrogenase... 

As indicated in Fig. 1, nitrogenase can reduce substrates other than Na. In the absence of other reducible substrates it will reduce protons to dihydrogen, but it can also reduce a number of other small triple-bonded substrates, as indicated in Section V,E,1. Large substrates are not reduced efficiently, indicating physical limitations on access to the enzyme s active site. CO is a potent inhibitor of all nitrogenase substrate reductions except that of the proton to Ha. In the presence of CO the rate of electron transfer is generally not inhibited, but all electrons go toward the production of Ha. 

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. 

Meyer, J. (1981). Comparison of carbon monoxide, nitric oxide, and nitrite as inhibitors of the nitrogenase from Cbstridium pasteurianum. Arch. Biochem. Biophys. 210, 246-256. 

Since the discovery by Cantley et al. that vanadate is a powerful and allosteric inhibitor of Na+,K+-ATPase,921 vanadium began to interest biochemists and Chasteen has reviewed work published up to 1983.471 Particularly interesting new studies involve vanadium peroxidases946 and nitrogenases.947... 

There are several circumstantial lines of evidence that indicate that N2 binds to the enzyme by displacing H2 (i.e., that the binding site of the enzyme is a metal hydride). The main evidence for a hydride site is (1) that H2 is a specific, competitive inhibitor for the reduction of N2 (2) the limiting stoichiometry of the MoFe nitrogenase in which we observe the apparent obligatory evolution of H2 during... 

What is known, though, is that in addition to its physiological substrates, that is, N2 and H+, nitrogenase is able to reduce a large variety of small doubly and triply bonded substrates (Table 2). Progress has been made in understanding the interaction of the FeMo cofactor site with such substrates or inhibitors as C2H2, CS2, or as well as that... 

ENDOR Studies on Inhibitor States of the Nitrogenase FeMo-cofactor—Carbon Monoxide... 

A major biochemical breakthrough in the biochemical investigation of nitrogenase was the ability to prepare EPR-active inhibitor states of the enzyme. Under traditional... 

Figure 10 35 GHz CW C ENDOR (except for Mims ENDOR as radicated) of lo-CO and hi-CO inhibitor forms of nitrogenase FeMo-cofactor, prepared with both CO and CO. The spectra were recorded at g ax of each species, as indicated. The spectra are centered...

From a biochemical point of view, the summation of the ENDOR studies on nitrogenase FeMo-cofactor interacting with carbon-containing small molecules is an electron inventory of the resting state, lo-CO form, and ethyne-bound form (Sepri), resulting in the proposal that the resting state is two electrons more oxidized than the substrate/inhibitor-bound forms and further, that these forms could be placed into the context of the Lowe-Thomeley scheme of nitrogen fixation. 

Inhibitors have been used not only to identify pathways of N metabolism but also to distinguish between uptake and assimilation of inorganic N. For example, MSX is an irreversible inhibitor of GS and has been used to explore the role of N assimilation products (e.g., gin) and intracellular NH4 on the feedback inhibition of processes such as nitrogenase activity and NOa uptake (Arp and Zumft, 1983). In addition, the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been used to distinguish between uptake and assimilation of NH4 in marine algae (Rees et al., 1998). 

Seefeldt L. C., Rasche M. E., and Ensign S. A. (1995) Carbonyl sulfide and carbon dioxide as new substrates, and carbon disulfide as a new inhibitor, of nitrogenase. Biochemistry 34, 5382-5389. 

It is potentially fruitful to pursue the intimate connection between H2 and the N2 binding site in nitrogenase. It has been shown unequivocallythat one H2 is evolved for each N2 fixed even at 50 atm of N2, a pressure of N2 well above full saturation. Moreover, H2 is a potent inhibitor of N2 fixation, and under D2, HD is formed, but only in the presence of N2. These complex relationships between N2 and H2(D2) have elicited a variety of interpretations. 

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