Preparation of Testis Non-Heme Iron Protein and Substitution for Adrenodoxin by Various Non-Heme Iron Proteins in Steroid 11-β-Hydroxylation

A highly purified non-heme iron protein has been prepared from pig testis. The protein contains iron and labile sulfide in approximately equimolar amounts. Optical absorption maxima occur at 455 mμ, 415 mμ, 320 mμ and 276 mμ with a small shoulder at 518 mμ in the oxidized form. In the reduced form, the absorption in the visible region is markedly bleached and a new maximum appears at 550 mμ. The optical rotatory dispersion and the circular dichroism spectra exhibit marked multiple Cotton effects. Upon reduction, the dispersion spectrum is greatly changed. The electron spin resonance spectrum exhibits a temperature sensitive signal at g=1.94 upon reduction. All of the properties are strikingly similar to those of adrenal non-heme iron protein (adrenodoxin) and plant ferredoxins. The substitution of the non-heme iron protein from testis for adrenodoxin in the adrenal steroid 11-β-hydroxylation reaction was accomplished, and the preparation of non-heme iron protein from ovary could also be substituted for adrenodoxin, whereas the similar preparation from liver, spinach and Euglena ferredoxins, Pseudomonas rubredoxin and putida redoxin were all inactive. The absence of adrenodoxin-like non-heme iron protein in the brain, liver, kidney and pancreas is suggested.