Formation and movement of Fe(III) in horse spleen, H- and L-recombinant ferritins. A fluorescence study.
Iron oxidation and incorporation into apoferritins of different subunit composition, namely the recombinant H and L homopolymers and the natural horse spleen heteropolymer (10-15% H), have been followed by steady-state and time-resolved fluorescence. After aerobic addition of 100 Fe(II) atoms/polymer, markedly different kinetic profiles are observed. In the rL-homopolymer a slow monotonic fluorescence quenching is observed which reflects binding, slow oxidation at the threefold apoferritin channels, and diffusion into the protein cavity. In the rH-homopolymer a fast fluorescence quenching is followed by a partial, slow recovery. The two processes have been attributed to Fe(II) binding and oxidation at the ferroxidase centers and to Fe(III) released into the cavity, respectively. The fluorescence kinetics of horse spleen apoferritin is dominated by the H chain contribution and resembles that of the H homopolymer. It brings out clearly that the rate of the overall process is limited by the rate at which Fe(III) leaves the ferroxidase centers of the H chains where binding of incoming Fe(II) and its oxidation take place. The data obtained upon stepwise addition of iron and the results of optical absorption measurements confirm this picture. The correspondence between steady-state and time-resolved data is remarkably good; this is manifest when the latter are used to calculate the change in fluorescence intensity as apparent in the steady-state measurements.
Cavallo
S
S
C.S. Biologia Molecolare, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Rome, Italy.
Mei
G
G
Stefanini
S
S
Rosato
N
N
Finazzi-Agrò
A
A
Chiancone
E
E
D000818:Animals; D001704:Biopolymers; D005293:Ferritins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006736:Horses; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000378:metabolism*
D001704:Biopolymers; D011994:Recombinant Proteins; D005293:Ferritins