$F_1F_0$ ATP synthase was prepared from iron-overloaded heart mitochondria to study the effect of iron on mitochondria. As $F_1F_0$ ATP synthase was able to transport iron, proteoliposomes containing $F_1F_0$ ATP synthase were prepared to compare iron uptake between control and iron-overloaded mitochondria. A threefold increase in $V_{max}$ (nmol/min/mg) ($6.35{pm}0.17$ vs. $2.08{pm}0.06$) and an eightfold increase in $K_m$ (${mu}M$) ($7.5{pm}0.7$ vs. $0.85{pm}0.5$) by $F_1F_0$ ATP synthase for iron uptake were observed in iron-overloaded mitochondria. Mitochondrial ATP synthase prepared from iron-overloaded heart has a canonical subunit composition in an altered stoichiometry compared to the control enzyme: the ${alpha}$, ${eta}$, ${gamma}$, OSCP, d, a, ${delta}$, and c subunits increased, but the b, e, A6L, and F6 subunits decreased significantly. In addition, the pattern of ${eta}$ subunit isomers with different pIs changed. These isomers appeared to be associated with augmented phosphorylation by excess iron.