PubMed1974
The Mo-Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in (57)Fe-enriched medium. The Mössbauer spectrum of the Mo-Fe protein, as isolated in the presence of Na(2)S(2)O(4), showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with delta=0.65mm/s and DeltaE=3.05mm/s at 4.2 degrees 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 delta=0.61mm/s and DeltaE=0.83mm/s at 77 degrees K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe(4)S(4) or Fe(2)S(2) low-spin ferrous iron clusters. Species M6, with delta=0.37mm/s and DeltaE=0.71mm/s at 77 degrees K, also corresponded to eight iron atoms/molecule of protein and, at 4.2 degrees 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 (delta=0.37mm/s, DeltaE=0.75mm/s at 77 degrees K, broad magnetic component at 4.2 degrees K) and M2 (delta=0.35mm/s, DeltaE=0.9mm/s at 4.2 degrees K). In the presence of the Fe protein, Na(2)S(2)O(4), ATP and Mg(2+), the M6 component of the Mo-Fe protein was replaced by species M7 with delta=0.46mm/s, DeltaE=1.04mm/s at 4.2 degrees 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, Na(2)S(2)O(4)-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had delta=0.50mm/s, DeltaE=0.9mm/s at 195 degrees K, and at 4.2 degrees 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 N(2) and C(2)H(2) and the inhibitor CO in the presence of ATP, Mg(2+) and Na(2)S(2)O(4). A number of Mössbauer spectral species associated with inactivated Mo-Fe and Fe proteins are described and discussed.