Clusters in Nitrogenase Proteins

Several spectroscopic techniques have been used to study nitrogenase proteins both in the isolated forms and during enzymatic activity. These include electron paramagnetic resonance (epr) spectroscopy, Mossbauer spectroscopy, uv-visible spectrophotometry, circular dichroism. X-ray absorption edge and fine structure spectroscopy (EXAFS), and linear electric field effects (LEFE). 

Attempts to study the nature of Fe in the MoFe protein have extended to oxidation studies on Avl and Kpl. From Mossbauer studies Orme-Johnson et al. (1977) defined four states of Fe in native dithionite-reduced Avl. These included (8 Fe/mol, Mg in Kpl) state D (9 Fe/mol equivalent to the epr silent 8-Fe group Mg in Kpl) Fe (3 Fe/mol equivalent to the 2-Fe group M4 in Kpl) and state S (1 Fe/mol) which may be an impurity (Munck et al., 1975). In a later study Zimmerman et al. (1978) modified these numbers they concluded that M, ccmtained 12 Fe in two apparently identical clusters. Species D and Fe were replaced by four P clusters each containing three irons of the D type and one of the Fe type. The remaining irons were in species S (2/mol) which, although inert under all conditions tested, were present in Cpl, Avl, and Kpl. Presumably, species S is not an impurity. The authors emphasized that active MoFe protein should contain 30 2 Fe/mol and that the P clusters, although spectroscopically unique, were probably closely related to conventional 4Fe4S clusters. 

Despite differences in nomenclature, experimental ctmditions, and protein (Kpl instead of Avl) Smith and Lang (1974) in their Mossbauer studies of 

In contrast to the other MoFe proteins, epr-monitored oxidation-reduction experiments with isolated Cvl yielded two 1-electron redox processes with midpoint potentials at -260 and 60 mV. Albrecht and Evans (1973) suggested that both centers were present in each molecule of Cvl however, O Donnell and Smith, to bring this observation in line with evidence from the redox titrations of the other MoFe proteins, argued that the two centers could arise from two different forms of the enzyme. 

redox titrations indicate that possibly three redox reactions are associated with the MoFe protein two with the isolated protein and one when it is associated with the Fe protein and ATP in the active nitrogenase. O Donnell and Smith aigue that the high redox potential step in the isolated protein has no role in enzyme activity because it is not conserved (i.e., the 

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