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A Mossbauer spectroscopic investigation of the redox behaviour of the molybdenum-iron protein from Klebsiella pneumoniae nitrogenase. Mechanistic and structural implications.

ABSTRACT: The redox properties of the nitrogenase Mo-Fe protein from Klebsiella pneumoniae have been monitored by 57Fe Mössbauer spectroscopy between -460 and -160mV (relative to the normal hydrogen electrode). Two redox processes associated with the atoms of the protein were observed. One at -216mV (pH 8.7) was associated with the Fe-Mo cofactor centres in the protein and allowed identification of the Mössbauer parameters of the oxidized form of these centres. The other redox process at -340mV (pH 8.7) was associated with species M5 [Smith & Lang (1974) Biochem. J. 137, 169-180]. This latter redox process may be involved in enzyme turnover. The oxidized form of species M5 interacts magnetically with species M4. The structural implications of the data have been considered in relation to other published data. It is concluded that an unequivocal assignment of the M4 and M5 atoms to Fe-S cluster types is not yet possible.

SUBMITTER: Smith BE

PROVIDER: S-EPMC1162235 | biostudies-other | 1980 Nov

REPOSITORIES: biostudies-other

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Project description: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.

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A Mossbauer spectroscopic investigation of the redox behaviour of the molybdenum-iron protein from Klebsiella pneumoniae nitrogenase. Mechanistic and structural implications.

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