Cobalt and desferrioxamine, like hypoxia, stimulate the production of erythropoietin in HepG2 cells. It is believed that cobalt as well as desferrioxamine interact with the central iron atom of heme proteins by changing their redox state similar to hypoxia. A subsequent decrease of the intracellular H2O2 levels under hypoxia was presumed to be the key event for stimulating erythropoietin production. We therefore investigated whether cobalt and desferrioxamine control the intracellular H2O2 levels that regulate gene expression by interacting with hemeproteins. Deconvolution of light absorption spectra revealed respiratory heme proteins such as cytochrome c, b558 and cytochrome aa3, as well as cytochrome b558, which is a nonrespiratory heme protein found in HepG2 cells. Whereas respiratory heme proteins are located in mitochondria, cytochrome b558 similar to the one described for the neutrophil NADPH oxidase can be visualized in the cell membrane of HepG2 cells by immunohistochemistry. Incubation with cobalt (100 microM/24 hr) interacts predominantly with cytochrome b558 and cytochrome b558. The interaction of cobalt with the respiratory chain results in an increased oxygen consumption of HepG2 cells as revealed by PO2 microelectrode measurements. Desferrioxamine (130 microM/24 hr), however has no influence on the cytochromes. In response to an external application of NADH (1 mM), the membrane bound cytochrome b558 produces two times more O2- than to the external NADPH (1 mM) application. Neither desferrioxamine not cobalt has any influence on the NADH stimulated O2- generation. Incubation with cobalt or with desferrioxamine, however, leads to a decrease of the intracellular H2O2 level as revealed by the dihydrorhodamine 123 technique, perhaps causing the well-known enhanced erythropoietin production. The cobalt-induced H2O2 decrease seems to be caused by an increased activity of the glutathion peroxidase that is also induced under hypoxia. Desferrioxamine, however, leads to an apparent H2O2 decrease only because it seems to inhibit the iron catalyzed reaction of H2O2 with dihydrorhodamine 123, hinting at the occurrence of the Fenton reaction in HepG2 cells. Therefore, it must be determined whether or not degradation products of H2O2 by the Fenton reaction suppress erythropoietin production under normoxia.