Dataset Information

Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae.

ABSTRACT: 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.

SUBMITTER: Smith BE

PROVIDER: S-EPMC1165827 | biostudies-other | 1973 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein-O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the S = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis.

Dataset Information

Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets