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Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins.

ABSTRACT: Stopped-flow kinetic data have been obtained for a rapid electron-transfer reaction between the component proteins of nitrogenase from Klebsiella pneumoniae, which was induced by MgATP. Up to three equivalents of the Fe-containing protein were rapidly oxidized by one equivalent of the Fe-Mo-containing protein in a unimolecular reaction, k2 = 2 x 10(2)S-1. Evidence for a tight complex between the component proteins, KD(complex) less than 0.5 muM, which was formed with a rate k1 greater than 1 x 10(7)M-1-S-1, has been obtained. MgATP bound to either the Fe-containing protein or to the two-protein complex with a rate k3 greater than 2.5 x 10(6)M-1-S-1 and with KD(MgATP) = 0.4mM, before the electron-transfer reaction.

SUBMITTER: Thorneley RN

PROVIDER: S-EPMC1165229 | biostudies-other | 1975 Feb

REPOSITORIES: biostudies-other

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Project description:The properties and catalytic reactions of the enzyme nitrogenase purified from Klebsiella pneumoniae were studied by electron-paramagnetic-resonance (e.p.r.) spectroscopy at temperatures down to 8 degrees K. The two protein fractions, Kp1 (the iron-molybdenum protein) and Kp2 (the iron protein), were examined alone and in steady-state mixtures and also in pre-steady-state experiments, by using the rapid-freezing method. Kp1 protein in dithionite solution shows a rhombic type of spectrum with g(1) 4.32, g(2) 3.63, g(3) 2.009 at pH6.8 (0 degrees C). Small changes in the spectrum produced by protons (pK=8.7 at 0 degrees C) or by acetylene indicate binding of these oxidizing substrates to this protein fraction. Kp2 protein shows a rhombic spectrum with g(1) 2.053, g(2) 1.942, g(3) 1.865, which integrates to about 0.45 electron/molecule. Binding of ATP, with a dissociation constant of 4x10(-4)m, changes the spectrum to an axial form with g( parallel) 2.036, g( perpendicular) 1.929, thus indicating a conformation change of Kp2 protein. The Kp2 protein spectrum disappears reversibly on cautious oxidation. The signals of both proteins are diminished in their steady-state mixtures, obtained in the presence of ATP and dithionite (with an ATP-generating system and Mg(2+) ions) and with protons, N(2) or acetylene as oxidizing substrate. At the same time as dithionite is consumed in such reactions, the Kp1 protein signal is gradually restored and the Kp2 protein signal diminishes to zero. In rapid-freezing experiments the signals from the two proteins decreased at indistinguishable rates (t((1/2)) about 10ms), then they remained constant. Results are interpreted in terms of a scheme in which reducing equivalents pass from dithionite to Kp2 protein, then, in an ATP-dependent reaction to Kp1 protein, this being finally reoxidized by N(2) or another oxidizing substrate. In this scheme Kp1 protein cycles between its signal-giving state and a very highly reduced signal-free state.

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Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins.

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