SPECTRUM AND IRON CONTENT OF PROTEIN B2 FROM RIBONUCLEOSIDE DIPHOSPHATE REDUCTASE

Protein B2, one of the two non‐identical subunits of ribonucleoside diphosphate reductase from Escherichia coli B, contains two atoms of inorganic iron per molecule of protein. The metal could be removed from the protein either by prolonged dialysis against 8‐hydroxyquinoline or by precipitation with acid ammonium sulphate. This resulted in loss of enzyme activity which was recovered after treatment of the apoprotein with Fe2+ or Fe3+ but not with any other metals tested. Removal of iron did not change the behaviour of the apoprotein on sucrose gradient centrifugation or on polyacrylamide gel electrophoresis as compared to native protein B2. No “labile sulphide” could be demonstrated in protein B2. Protein B2 had a characteristic spectrum with a very sharp peak at 410 nm, a broader one around 360 nm and a steep shoulder at approximately 325 nm. The two peaks disappeared on removal of iron and reappeared on reactivation of the enzyme with the metal. When protein B2 was treated with hydroxyurea or hydroxylamine the peak at 410 nm (but not that at 360 nm) disappeared in parallel with enzyme activity. Such treatment did not result in the removal of iron. It is suggested that the very sharp peak at 410 nm originates from an unidentified component of protein B2. Furthermore, this component, together with iron, may be the functional equivalent in the E. coli system of cobamide coenzyme in ribonucleoside triphosphate reductase of Lacto‐bacillus leichmannii. Copyright © 1969, Wiley Blackwell. All rights reserved