MOSSBAUER AND INTEGER-SPIN EPR OF THE OXIDIZED P-CLUSTERS OF NITROGENASE - POX IS A NON-KRAMERS SYSTEM WITH A NEARLY DEGENERATE GROUND DOUBLET

The molybdenum-iron protein of nitrogenase contains 2 Mo atoms and ca. 28-30 Fe atoms. Approximately 16 Fe atoms belong to the P-clusters, a novel type of iron-sulfur cluster of unknown structure. Mossbauer studies have established that P-clusters are diamagnetic in the semireduced state, P(N). Upon oxidation of the protein with redox dyes such as thionin the state P(OX) is attained. Previous studies have revealed that the low-temperature (less-than-or-equal-to 4.2 K) Mossbauer spectra of P(OX) exhibit magnetic hyperfine patterns even in the absence of external magnetic fields. Such behavior is generally characteristic of a Kramers system, i.e., of a system with an odd number of electrons. The spectra had features typical of those observed for a Kramers doublet with extremely anisotropic g-values (g1 >> g2, g3). Recent Mossbauer and EPR studies of integer spin systems in our laboratory have suggested the possibility that P(OX) may be a very unusual non-Kramers system. Here we report Mossbauer and EPR studies of the proteins from Azotobacter vinelandii (Av1), Clostridium pasteurianum (Cp1), Klebsiella pneumoniae (Kp1), and Xanthobacter autotrophicus (Xa1) which prove that the electronic ground doublet of P(OX) is not a Kramers doublet but rather a nearly degenerate doublet (splitting DELTA) of a system with an even number of electrons. Cp1, Av1, and Kp1 have DELTA less-than-or-equal-to 10(-3) cm-1, and the magnetic patterns observed in the zero-field Mossbauer spectra result from mixing of the two electronic levels by Fe-57 hyperfine interactions (\A(z)\ almost-equal-to 10(-3) cm-1). For Av1 and Kp1 we have observed integer-spin EPR transitions, at g(eff) = 11.9, between two excited-state spin levels at 10-15 cm-1. The ground doublet of Xa1 exhibits an integer-spin resonance at g(eff) = 15.6. Analysis of the Xa1 Mossbauer spectra yields DELTA almost-equal-to 0.010 cm-1. Using this value of DELTA, quantitation of the EPR spectra yielded ca. two spins per MoFe protein for P(OX). The observed g-values suggested that P(OX) of Xa1 has S = 3 or S = 4. However, the ground- and excited-state DELTA-values of Kp1 and Av1 indicate that the electronic ground manifold may not consist of an isolated multiplet with definite spin S. Recognition of P(OX) as a non-Kramers state implies that two electrons are removed from each P-cluster in the transformation P(N) --> P(OX). Since ca. four electrons per MoFe protein are removed and since ca. 16 Fe atoms are involved, it follows that the alphabeta dimer of nitrogenase contains two identical P-clusters and that each cluster has ca. 8 Fe sites. The Mossbauer data are reviewed for a model that considers the P-clusters to consist of two bridged cubanes.