Iron release in vitro from human serum diferric transferrin (hFe(2)TO in acidic media (4.2 <= pH <= 5.4) in the presence of nonsynergistic anions occurs in at least five kinetic steps. Step 1 (most rapid) involves proton assisted release of carbonate from the protein. In subsequent steps, iron release from both the N- and C-terminal lobes is controlled by slow proton transfers and anion binding. In step 2, the N-terminal lobe takes up one proton with kinetic linkage to the binding of one anion. In step 3, iron release from the anion-linked N-terminal lobe is controlled by slow uptake of two protons with rate-constant, k(2N), of 2.6(6) x 107, 6.1(6) x 10, and 9(1) x 10(7) M-2 s(-1) in the presence of Cl-, NO3-, and SO42-, respectively. In step 4, the C-terminal lobe takes up one proton with kinetic linkage to the binding of one anion. In step 5, iron release from the anion-linked C-terminal lobe is controlled by slow uptake of two protons with rate-constant, k(2C), of 8.4(2) X 10(4), 4.4(6) X 10(5), and 8.1(2) x 10(5) M-2 s(-1) in the presence of Cl-, NO3-, and SO42-, respectively.