MODULATION OF THE REDOX STATE OF THE COPPER SITES OF HUMAN CERULOPLASMIN BY CHLORIDE

Incubation of human ceruloplasmin with physiological concentrations of chloride at neutral pH invariably caused dramatic changes of both the spectroscopic and the functional properties of the protein. The optic al intensity at 610 nm increased up to 60%, with a concomitant decreas e at 330 nm and the appearance of new bands between 410 and 500 nm, Si gnals previously undetectable appeared in the EPR spectrum. On the bas is of computer simulations, they were interpreted as stemming from an oxidized type 1 copper site and from a half-reduced type 3 copper pair . Removal of chloride completely restored the original optical and EPR lineshapes. Hydrogen peroxide, added to ceruloplasmin in the presence of chloride, was able to capture the electron of the half-reduced typ e 3 site and to yield a protein insensitive to subsequent removal and readdition of the anion. As a whole, the spectroscopic data indicate t hat a blue site is partially reduced in the resting protein and that, upon binding of chloride, human ceruloplasmin undergoes a structural c hange leading to displacement of an electron from the reduced type 1 s ite to the type 3 site pair. Chloride dramatically affected the cataly tic efficiency of human ceruloplasmin. At neutral pH, the anion was an activator of the oxidase activity, being able to enhance up to tenfol d the catalytic rate. At pH < 6, in line with all previous reports, ch loride strongly inhibited the activity, At intermediate pH values, i.e ., around 6, the effect was composite, with an activating effect at lo w concentration and an inhibitory effect at higher concentration. Sinc e chloride is present at very high concentrations in the plasma, these results suggest that human ceruloplasmin is, in the plasma, under con trol of this anion.