Cytochrome aa₃ from Nitrosomonas europaea

Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X-100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm^-1 mM^-1. The enzyme has ~11 nmol of heme a and ~11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a₃), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa₃-type oxidase. Although the enzyme had been previously classified as 'cytochrome a₁' on the basis of its ferrous α absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa₃. Neither the data reported here nor a review of the literature supports the existence of cytochrome a₁ as an entity discrete from cytochrome aa₃. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo.