Mutations in the Ca²⁺ binding site of the Paracoccus denitrificans cytochrome c oxidase

Abstract

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial sitedirected mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca 2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca 2+ ligand. z 1999 Federation of European Biochemical Societies.

Coupling between protonation and conformation in cytochrome c oxidase: Insights from constant-pH MD simulations

“…It should be noted that none of our systems contains the crystallographic calcium ion bound close to the surface of the protein [6,8]. It was shown experimentally that calcium ions do not affect the activity of bacterial CcO [42,43] nor induce spectral shift of heme a [44,45].…”

Ca2+-Binding Site in Rhodobacter Sphaeroides Cytochrome c Oxidase

“…Mutants in the Ca 2+ -binding site of the P. denitrificans COX have been examined by two groups (25,39). The results are not in total agreement.…”

Cytochrome c oxidase — structure, function, and physiology of a redox-driven molecular machine

“…It should be noted at this point that attempts to crystallize oxidase(s) in different redox states (Yoshikawa et al 1998;Harrenga and Michel 1999) have not provided any evidence that major structural differences accompany the (complete) reduction of the enzyme cofactors. In the Paracoccus oxidase, the binuclear center conformation remains virtually unchanged, with a distance between metal ions of 5.2 and all three Cu B -liganding histidines in place in both redox states.…”

“…The four cytochrome c oxidase structures Tsukihara et al 1995Tsukihara et al , 1996Ostermeier et al 1997;Yoshikawa et al 1998;Harrenga and Michel 1999;Soulimane et al 2000;Svensson-Ek et al 2002) reveal a striking degree of identity among each other, irrespective of the fact that the mitochondrial enzyme is the product of two separate genetic systems and displays a tremendous increase of subunit complexity. The enzymes from Paracoccus denitrificans and Rhodobacter sphaeroides may be viewed as simple bacterial model systems that perfectly match the mitochondrial enzyme, at least in all the basic enzymatic functions, while the Thermus thermophilus oxidase appears more distant in terms of characteristic common structural and functional signatures (see below).…”

“…The copper ion is liganded by three histidines, both in the oxidized and the reduced forms of the enzyme (Harrenga and Michel 1999), and has been determined to be 5.2 away from the heme a 3 iron in the four-subunit enzyme, and 4.5 in the two-subunit complex (with a different pH used in the crystallization buffer for both preparations).…”