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Metabolism and Enzymology

Formation of the Fe-S Cluster of Ferredoxin in Lysed Spinach Chloroplasts ¹

Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan

In vitro formation of the ³⁵S-labeled Fe-S cluster of ferredoxin (Fd) has been achieved by incubating apo-Fd and [³⁵S]cysteine with osmotically lysed chloroplasts of spinach (Spinacia oleracea). Correct integration of the ³⁵S-labeled Fe-S cluster into Fd was verified on the basis of the following: (a) Under nondenaturing conditions, ³⁵S-labeled holo-Fd showed the same electrophoretic mobility as authentic holo-Fd; (b) ³⁵S-labeled holo-Fd showed an ability to bind Fd-NADP⁺ reductase; (c) the ³⁵S-labeled moiety was removed from the Fd polypeptide by TCA treatment but not by 2-mercaptoethanol treatment; (d) externally added pea II apo-Fd was converted to ³⁵S-labeled holo-Fd. This reconstitution was dependent on both ATP and light, and formation of the ³⁵S-labeled Fe-S cluster was observed upon addition of ATP or when an ATP generation-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substitute, indicating the requirement of ATP hydrolysis for cluster formation. GTP was able to substitute for ATP, but CTP and UTP were less effective. Fe-S cluster formation in lysed chloroplasts was stimulated by light even in the presence of added ATP. Light stimulation was inhibited by DCMU or methyl viologen but not by NH₄⁺. NADPH was able to substitute for light, indicating that light energy is required for the production of reducing compounds such as NADPH in addition to the generation of ATP. These results confirm the requirement of light for the Fe-S cluster formation observed previously in intact chloroplasts.

¹ This work was supported in part by Grants-in-Aid for Encouragement of Young Scientists (No. 61740389 and 62740390) from the Ministry of Education, Science and Culture of Japan.

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