Microorganisms capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Phylogenetically distinct organisms were shown to express spectrally distinct redox-active biomolecules during autotrophic growth on soluble iron. A new iron-oxidizing eubacterium, designated as strain Funis, was investigated Strain Funis was judged to be different from other known iron-oxidizing bacteria on the bases of comparative lipid analyses, 16S rRNA sequence analyses, and cytochrome composition studies. When grown autotrophically on ferrous ions, Funis produced conspicuous levels of a novel acid-stable, acid-soluble yellow cytochrome with a distinctive absorbance peak at 579 nm in the reduced state. Stopped-flow spectrophotometric kinetic studies were conducted on respiratory chain components isolated from cell-free extracts of Thiobacillus ferrooxidans. Experimental results were consistent with a model where the primary oxidant of ferrous ions is a highly aggregated c-type cytochrome that then reduces the periplasmic rusticyanin. The Fe(II)-dependent, cytochrome c-catalyzed reduction of the rusticyanin possessed three kinetic properties in common with corresponding intact cells that respire on iron: the same anion specificity, a similar dependence of the rate on the concentration of ferrous ions, and similar rates at saturating concentrations of ferrous ions.