Molybdenum insertion in vitro in demolybdo nitrate reductase of Chlorella vulgaris

Demolybdo nitrate reductase (also called cyt c reductase) of Chlorella vulgaris has been converted to active nitrate reductase by insertion of Mo from Na2MoO4 in vitro. A procedure is described which consistently gives about 0.3 unit of nitrate reductase from about 6 units of cyt c reductase, a yield of 30% of the maximum expected, if we calculate on a basis of a ratio of 6 to 1 for the cyt c reductase/nitrate reductase of purified normal enzyme. The demolybdoenzyme is incubated for 30 s at 31 degrees C with molybdate and reduced glutathione (GSH) at pH 4.8, and the pH is then raised to 7, and the incubation continued for 20 min. At the acid pH, there must be a partial denaturation or unfolding which permits Mo insertion, with a refolding to active enzyme at the higher pH. The GSH is not essential for activation, but in its absence the yield of active enzyme was about 50% lower. Experiments with labeled GSH showed that no GSH was incorporated into the protein during the activation procedure. Although the enzyme activity measurements suggested that only 30% of the enzyme was activated, measurements with 99Mo showed that there was one Mo incorporated per subunit weight of 90,000. The Km for nitrate of the activated nitrate reductase was identical with the Km for nitrate of the normal enzyme. On gradient centrifugation, activated nitrate reductase, cyt c reductase, and normal nitrate reductase all behaved identically.