The introduction of unsaturation into steroid ring A is of interest in connection with the aromatization involved in the biosynthesis of phenolic estrogens and the enhanced physiological activity of A'-dehydrosteroids. Many microbial species carry out the removal of hydrogen atoms from adjacent carbon atoms at C-i(2) and C-4(s) to yield the corresponding A1- and A4-dehydrosteroids (166). During the course of studies on the oxidative degradation of steroids by P. testosteroni, it was found that this micro-organism was capable of effecting such dehydrogenations and of converting 5a-androstane-3,i7-dione and 50-androstane-3,i7-dione to i,4-androstadiene-3,i7-dione. The same system also transformed 19-nor steroids to the corresponding ring A phenols (133).

The dehydrogenations of carbon to carbon bonds in steroids have been demonstrated with partially purified enzyme preparations, and the detaded mechanisms of these reactions investigated (133, 134, 135). The enzymes responsible for these transformations in P. testosteroni (Δ-dehydrogenases) are steroid-induced. They are firmly associated with small intracellular particles which are sedimented only after prolonged centrifugation at 100,000 X g. Whereas crude bacterial sonicates carry out Δ-dehydrogena-tions rapidly and efficiently without further additions, the enzyme preparations are rendered inactive upon centrifugation at 20,000 X g. Full activity is restored by the addition of phenazine methosulfate (PMS), an artificial electron acceptor which can substitute for the unidentified natural acceptor. Of many electron acceptors examined, PMS was the only one found to be active in this system.

Several methods have been devised for the assay of Δ-dehydrogenases. The formation of ring A-phenolic steroids from ig-nor compounds serves as a sensitive assay. The reactions may also be studied by observing the bleaching of PMS which accompanies the reduction of the dye under anaerobic conditions. More conveniendy, the reduction of PMS may be coupled to the non-enzymatic reduction of cytochrome c under aerobic conditions, since reduced PMS reacts with cytochrome c in preference to oxygen (Massey, V. Biochim. et biophys. acta, 34:255, 1959).

The Δ-dehydrogenases exhibit both steric and positional specificity for their steroidal substrates. In P. testosteroni, three distinct A dehydrogenases have been recognized (133, 134, 135).Δ1-Dehydrogenase promotes the removal of a pair of hydrogen atoms from positions 1 and 2 of various 5α-androstane, 5β-androstane and estrane derivatives, for example:

4-Androstene-3,17-dione + PMS→1,4-androstadiene-3,17-dione + PMSH2 ;

4-Estrene-3,17-dione + PMS → estrone + PMSH2.

Δ4-(5 α-Dehydrogenase catalyzes the introduction of double bonds into the 4-5 position of C,8 and C,9 steroids in which the A and B rings are fused trans. Typical reactions catalyzed by this enzyme are the following: 1-(5a)-Androstene-3,17-dione + PMS→ 1,4-androstadiene-3,17-dione + PMSHi; 1-(5α)-Estrene-3,17-dione + PMS → estrone + PMSHS.

Both Δ1- and Δ4-(5 α-dehydrogenases have been partially purified from P. testosteroni, and the influence of structural alterations in the substrate on the reaction velocity has been examined. A separate Δ4-5β-dehydrogenase is concerned with removal of a pair of hydrogen atoms from the 4 and 5β positions.

A-Dehydrogenations can occur anaerobically. Various steroids oxygenated in the C-1 and C-2 positions cannot serve as substrates for Δ1-dehydrogenase. For these and other reasons, it has been concluded that ΔA-dehydrogenases act through a direct and irreversible abstraction of hydrogen atoms from adjacent carbon atoms on the steroid skeleton and that the reactions do not involve hydroxylated intermediates. The nature of the acceptor group on the enzyme is not known, but some evidence suggests that this may be a flavin (134, 135).