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Mossbauer 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 (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.

SUBMITTER: Smith BE

PROVIDER: S-EPMC1166102 | biostudies-other | 1974 Feb

REPOSITORIES: biostudies-other

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Project description:1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s(0) (20,w)=11.0S and D(0) (20,w)=4.94x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H(2) evolution, 380 for N(2) reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s(0) (20,w)=4.8S and D(0) (20,w)=5.55x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H(2) evolution, 275 for N(2) reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77 degrees K. The u.v. spectra of reduced and O(2)-inactivated proteins were identical, and inactivation by O(2) generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O(2) decreased the depth of the trough. 5. The reduction of N(2) and acetylene, and H(2) evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo-Fe-containing protein; excess of the Mo-Fe-containing protein was inhibitory. All reductions were accompanied by H(2) evolution. The combined proteins had no ATP-independent hydrogenase activity.

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

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