Upon separation of a crude preparation of beta subunit ("beta fraction") from mitochondrial F-1-ATPase containing one equivalent of Fe(III) in the nuelcotide-independent site (1Fe(III)-loaded MF1), Fe(III) is almost completely recovered. CD spectra show that "beta fraction" maintains the structural changes induced by Fe(III) in the whole enzyme. In accordance, EPR reveals that the Fe(III) site geometry is conserved in "beta fraction." Moreover, the EPR spectra of 1Fe(III)-loaded MF1 and its "beta fraction" undergo similar changes of the line-shape upon Pi binding at the catalytic site, indicating that the Pi and Fe(III) are proximal on P. Highly purified P in nucleotide-free form binds I mol of Fe(III)/mol of protein. MF1 "freezed" by inhibitors with two P in closed conformation and one P in open or half-closed conformation binds I mol of Fe(III)/mol of enzyme. Therefore, the Fe(III) site location in the unique P subunit not adopting the closed conformation is proposed. (C) 2002 Elsevier Science (USA). All rights reserved.