Mössbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions

Abstract

The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in ⁵⁷Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na₂S₂O₄, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe₄S₄ or Fe₂S₂ low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth's Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na₂S₂O₄, ATP and Mg²⁺, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na₂S₂O₄-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo–Fe and the Fe protein under argon were unaffected by the reducible substrates N₂ and C₂H₂ and the inhibitor CO in the presence of ATP, Mg²⁺ and Na₂S₂O₄. A number of Mössbauer spectral species associated with inactivated Mo–Fe and Fe proteins are described and discussed.