ZN-EXCHANGE AND MOSSBAUER STUDIES ON THE [FE-FE] DERIVATIVES OF THE PURPLE ACID FE(III)-ZN(II)-PHOSPHATASE FROM KIDNEY BEANS

In order to perform Mossbauer studies, Zn(II) in the Fe(III)-Zn(II) pu rple acid phosphatase of the red kidney bean has been exchanged by inc ubating the semiapoenzyme with Fe-57(II). The resulting Fe(III)-Fe-57( II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H(2)0(2) or peroxydisulfate to the Fe(III)-Fe-5 7(III) species with a 30-times lower activity. Incubation of the metal -free apoenzyme with Fe-57(II) in the presence of O2 leads to the Fe-5 7(III)-Fe-57(II) species which is stable in dilute solutions, but part ially oxidized during the concentration procedure to the Fe-57(III)-Fe -57(III) enzyme. Limited reduction of the oxidized enzyme with ascorba te delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, bu t not the mixed valent Fe(III)-Fe(II) species, indicating that after t he transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The Mossbau er spectra of the oxidized species show at 4.2 K two quadrupole double ts with delta of 0.51 mm/s and 0.53 mm/s and DELTAE of 1.46 and 0.96 m m/s indicating high spin Fe(III) in two different binding sites, obvio usly with a higher asymmetry in the chromophoric Fe(III) site. The val ues are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) s pecies shows two quadrupole doublets with delta values of 0.55 mm/s an d 1.14 mm/s and DELTAE values of 1.43 mm/s and 3.01 mm/s at 70 K for h igh spin Fe(II) and Fe(III), but the signal of the Fe(II) component sh ows magnetic patterns at 4.2 K indicating a half-integer spin system w ith antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and w ith DELTAE values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Additi on of phosphate shows only minor differences in the oxidized enzyme an d in the mixed valent Fe(III)-Fe(II) system. Interaction with O2 is di scussed.