Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.
Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)
Musci
G
G
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
Di Marco
S
S
Bonaccorsi di Patti
M C
MC
Calabrese
L
L
D000818:Animals; D002570:Ceruloplasmin / Q000737:chemistry* / Q000187:drug effects; D002645:Chickens; D003300:Copper / Q000737:chemistry*; D004347:Drug Interactions; D004578:Electron Spin Resonance Spectroscopy; D009569:Nitric Oxide / Q000737:chemistry* / Q000494:pharmacology; D010084:Oxidation-Reduction; D012756:Sheep
D009569:Nitric Oxide; D003300:Copper; D002570:Ceruloplasmin