Reduction of adenosine 5'-phosphosulfate to cysteine in extracts from Chlorella and mutants blocked for sulfate reduction.

Abstract

Techniques for preparation of enzyme extracts from Chlorella pyrenoidosa chick are described which yield a stable system which converts adenosine 5′‐phosphosulfate to sulfide and/or cysteine in the absence of added active thiols. Since these thiols (such as dithiothreitol) have been shown to yield sulfite, among other products, from the adenosine‐5′‐phosphosulfate sulfotransferase reaction, we have studied the adenosine 5′‐phosphusulfate reducing system in the presence and absence of dithiothreitol. In all cases dithiothreitol increases the activity of the reducing system forming cysteine from adenosine 5′‐phosphosulfate. Mutant Sat−2, which cannot grow on sulfate, is shown to lack thiosulfonate reductase activity but to retain sulfite reductase activity. Extracts from this mutant will form sulfide and cysteine only in the presence of dithiothreitol, while wild‐type extracts will form these products in presence or absence of dithiothreitol, the activity with dithiothreitol being higher. Mutants Sat−1,3–6 which are blocked in the adenosine‐5′‐phosphosulfate sulfotransferase step are blocked in the formation of sulfide and cysteine from adenosine 5′‐phosphosulfate. Binding studies with adenosine 5′‐phosphosulfate indicate that the sulfonyl group is transferred to a carrier to form carrier‐S‐SO−3 and the available evidence suggests that this carrier is a prosthetic group of the thiosulfonate reductase. On the basis of this evidence, two pathways in the cell‐free extracts are thought to be operating. One, in the absence of active thiols, is a pathway consisting of bound intermediates in which the sulfonyl group of adenosine 5′‐phosphosulfate is transferred via adenosine‐5′‐phosphosulfate sulfotransferase to carrier on the thiosulfonate reductase where it is reduced to the thiol level by ferredoxin; the thiol is then transferred via O‐acetyl‐l‐serine sulfhydrylase to form cysteine. The other, or free intermediate pathway operates in the presence of dithiothreitol which releases free sulfite from the adenosine‐5′‐phosphosulfate sulfotransferase reaction. This sulfite is then reduced via sulfite reductase to sulfide which forms cysteine via O‐acetyl‐l‐serine sulfhydrylase. Since mutant Sat−2, which cannot grow on sulfate nor reduce it in vivo, lacks thiosulfonate reductase activity but yields sulfite reductase activity in extracts, we conclude that the bound intermediate pathway is the preferred pathway of sulfate reduction in vivo; the free intermediate pathway is probably operative only when free sulfite is available either from occasional side reactions in vivo or when sulfite is fed to cells from outside.