This study was undertaken to characterize the nitric oxide complexes o
f mammalian ferritin and their EPR properties to gain a better underst
anding of the interaction of NO with non-heme iron proteins with in th
e cell. Measurements were made with horse spleen apo-and holoferritins
, with chemically modified proteins, and with recombinant human H-chai
n apoferritin and its site-directed mutants. Three types of EPR signal
s (A, B, and C) have been identified and attributed to iron-nitrosyl c
omplexes at imidazole groups of histidine, thiol groups of cysteine, a
nd carboxylate groups of aspartate and glutamate, respectively. The C-
type axial spectrum has features at g(perpendicular-to)' = 4 and g(par
allel-to)' = 2 characteristic of a paramagnetic Fe3+-NO- complex with
total spin S = 3/2 and probably arises from nonspecific binding to car
boxylate groups on the protein. The S = 1/2 axial B-type signal (g(per
pendicular-to)' = 2.033 and g(parallel-to)' = 2.014) is formed at Cys-
130 (human H-chain sequence numbering). His-128 and possibly His-118 a
re sites of formation of the rhombic S = 1/2 A-type complex (g(x)' = 2
.055, g(y)' = 2.033, and g(z)' = 2.015); the former residue perhaps pl
ays a role in the conformational stability of the protein as well as i
n iron binding. The data reveal that the residues Cys-130 and His-128
in the vicinity of 3-fold channels leading to the interior of the prot
ein shell are important in iron-nitrosyl complex formation in mammalia
n ferritins.