The calcium binding site in cytochrome aa(3) from Paracoccus denitrificans

A Shift in the spectrum of heme a induced by calcium or proton binding, or by the proton electrochemical gradient, has been attributed to interaction of Ca2+ or H+ with the vicinity of the heme propionates in mitochondrial cy tochrome c oxidase, and proposed to be associated with the exit path of pro ton translocation. However, this shift is absent in cytochrome c oxidases f rom yeast and bacteria [Kirichenko et al. (1998) FEES Lett. 423, 329-333]. Here we report that mutations of Glu56 or Gln63 in a newly described Ca2+/N a+ binding site in subunit I of cytochrome c oxidase from Paracoccus denitr ificans [Ostermeier et al. (1997) Proc. Natl. Acad Sci. U.S.A. 94, 10547-10 553] establish the Ca2+-dependent spectral shift in heme a. This shift is c ounteracted by low pH and by sodium ions, as was described for mammalian cy tochrome c oxidase, but in the mutant Paracoccus enzymes Na+ is also able t o shift the heme a spectrum, albeit to a smaller extent. We conclude that t he Ca2+-induced shift in both Paracoccus and mitochondrial cytochrome aa(3) is due to binding of the cation to the new metal binding site. Comparison of the structures of this site in the two types of enzyme allows rationaliz ation of their different reactivity with cations. Structural analysis and d ata from site-directed mutagenesis experiments suggest mechanisms by which the cation binding may influence the heme spectrum.