A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed ‘non-heme non-FeS iron’, is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K.

The ‘non-heme non-FeS iron’, which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 ± 0.3 nmol · mg⁻¹ protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 ± 0.2 nmol · mg⁻ protein) is localized to the ‘outer’ compartment and a major fraction (i.e. 1.1 ± 0.1 nmol · mg⁻¹ protein) is localized to the ‘inner’ compartment and is equally distributed between the inner membrane and the matrix.

The experiments described in this study also indicate that approximately half of the ‘non-heme non-FeS iron’ of the ‘inner’ pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.