alexa Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain.


Mycobacterial Diseases

Author(s): Rapisarda VA, Montelongo LR, Faras RN, Massa EM

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Abstract Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50\%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties. Copyright 1999 Academic Press. This article was published in Arch Biochem Biophys and referenced in Mycobacterial Diseases

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