Enzymic synthesis of the 4Fe–4S clusters of Clostridium pasteurianum ferredoxin

Abstract

Ex novo enzymic synthesis of the two 4Fe–4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, dl‐dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide. A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe–2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. (1) The nature of the cluster to be inserted into the reconstituted iron‐sulfur protein is determined by the apoprotein itself. (2) The refolding of the structure of the iron‐sulfur proteins around the newly inserted cluster is the rate‐limiting step in both chemical and enzymic reconstitution. (3) Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron‐sulfur protein(s). The extension to the 4Fe–4S centers of the rhodanese‐based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron‐sulfur structures. Copyright © 1985, Wiley Blackwell. All rights reserved