Abstract
Purified beef heart cytochrome c oxidase is inactivated to the extent of 35 to 50% by the nonpolar mercurial reagents mercuric chloride and ethylmercuric chloride. The inactivation is complete within 5 min. In titrations of activity, the plateau level of inactivation is attained at added ethylmercuric chloride:heme a ratios of about 1:1. Up to 3 mercury atoms/heme a are bound to the oxidase, although only the first of these affects its enzymatic activity. Incubation of the ethylmercury-modified oxidase with sulfhydryl compounds reverses the inactivation, with 2,3-dimercaptopropanol being most effective of the reagents tested. Spectrophotometric and polarographic assays of enzymatic activity show that Km values for the native and the ethylmercury-modified enzymes are practically indistinguishable, and that the partial inactivation observed for the latter is reflected exclusively in a lower value of Vmax compared to that of the native enzyme. Based on these results, we propose that ethylmercuric chloride reacts with a single crucial--SH group per heme a, and that electron transfer processes in the modified product are partially inhibited.
Highlights
Cytochrome c oxidase (EC 1.9.3.1) hasbeen shownby amino acid analysis to contain 7 cysteinyl residues out of a total of about 720 to 820 amino acid residues/heme a [1,2]
In crude extractsof cytochrome oxidase, for example,oxidase activity was inhibited 5 times more effectively by basic phenylmercuric nitrate than by pCMB [6].Incubation of cytochrome oxidase with hydrophobic copper chelators inhibits activity by 40 to 75% and changes the characteristic spectral features attributed to functional copper; the hydrophilic analogs, do notaffect the enzyme[16]
In order to assess the functional role of those sulfhydryl groups that may be inaccessible to hydrophilic reagents, we have studied thme odification of sulfhydryl groups of purified cytochromeoxidase by hydrophobic mercurials.Mercuric chloride is a relativelynonpolar substance andoes not ionize readily
Summary
From the DeDartments of Biochemistrv and o f Radiation Biology a n d Biophysics, The University of Rochester Schoolof Medicine, Rochester, New York 14642“. Purified beef heart cytochrome c oxidase is inactivated to the extent of 35 to 50% by the nonpolar mercurial reagents mercuric chloride and ethylmercuric chloride. Spectrophotometric and polarographic assays of enzymatic activity show that K,,, valuesfor the native and the ethylmercury-modified enzymes are practically indistinguishable, and that the partial inactivation observed f o r the latter is reflected exclusively in a lower value of V,, compared to that of the native enzyme Based on these results, we propose that ethylmercuric chloride reacts with a single crucial ”SH group per heme a,and that electron transfer processes in the modified product are partially inhibited. 5 pl of the mercurial modifying reagent of the desired concentration, dissolved in absolute ethanol, were added and the mixture was incubated at 25°C.Atspecified time intervals, aliquots were removed and assayed for activity. Large changes in conformationor state of aggregation may be ruled ouat s possible reasons for the partial inhibition obtained upon modification
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