Reversible, non-denaturing metal substitution in bovine adrenodoxin and spinach ferredoxin and the different reactivities of [2Fe-2S]-cluster-containing proteins.
The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.
Iametti
S
S
Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy.
Uhlmann
H
H
Sala
N
N
Bernhardt
R
R
Ragg
E
E
Bonomi
F
F
D000325:Adrenodoxin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry / Q000494:pharmacology; D002413:Cations, Divalent / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008670:Metals / Q000737:chemistry / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D018724:Spinacia oleracea
C081714:2Fe-2S ferredoxin; D002413:Cations, Divalent; D005288:Ferredoxins; D008670:Metals; D002104:Cadmium; D000325:Adrenodoxin