Point amino acid substitutions at Ca2+-binding sites of recoverin. I. The mechanism of successive filling of the Ca2+-binding sites

The molecule of photoreceptor Ca2+-binding protein recoverin contains four potential Ca2+-binding sites of the EF-hand type, but only two of them (the second and the third) can actually bind calcium ions. We studied the inter action of Ca2+ with recoverin and its mutant forms containing point amino a cid substitutions at the working Ca2+-binding sites by measuring the intrin sic protein fluorescence and found that the substitution of Gln for Glu res idues chelating Ca2+ in one (the second or the third) or simultaneously in bath (the second and the third) Ca2+-binding sites changes the affinity of the protein to Ca2+ ions in different ways. The Gln for Glu121 substitution in the third site and the simultaneous Gln substitutions in the second (fo r Glu85) and in the third (for Glu121) sites result in the complete loss of the capability of recoverin for a strong binding of Ca2+-ions. On the othe r hand, the Gln for Glu85 substitution only in the second site moderately a ffects its affinity to the cation. Hence, we assumed that recoverin success ively binds Ca2+-ions: the second site is filled with the cation only after the third site has been filed. The binding constants for the third and the second Ca2+-binding sites of recoverin determined by spectrofluorimetric t itration are 3.7 x 10(6) and 3.1 x 10(5) M-1, respectively.