Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca2+ ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca2+ reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca2+ coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca2+; their spectra are not perturbed by added Ca2+ or EGTA. The D485A mutant is active, binds to Ca2+ reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca2+ binding for both reduced and oxidized states of heme a. The Kd value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (Kd = 1-3 μM). The rate of Ca2+ binding with the D485A oxidase (kon = 5 × 103 M-1 s-1) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (∼10-3 s-1) and highly temperature-dependent. The koff /kon ratio (190 nM) is about 30-fold higher than the equilibrium Kd of 6 nM, indicating that formation of the Ca2+-adduct may involve more than one step. Sodium ions reverse the Ca2+-induced red shift of heme a and dramatically decrease the rate of Ca2+ binding to the D485A mutant COX. With the D485A mutant, 1 Ca2+ competes with 1 Na+ for the binding site, whereas 2 Na+ compete with 1 Ca2+ for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na+ binding site in bovine COX. No effect of Ca2+ binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca2+ plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.
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