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Evidence for the Sacrificial Role of the Auxiliary [4Fe‐4S] Cluster of Lipoyl Synthase
Author(s) -
Lanz Nicholas,
Pandelia MariaEirini,
McLaughlin Martin,
Rectenwald Justin,
Silakov Alexey,
Drennan Catherine,
Krebs Carsten,
Booker Squire
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.4
Subject(s) - chemistry , sulfur , linker , cluster (spacecraft) , iron–sulfur cluster , crystallography , substrate (aquarium) , stereochemistry , cofactor , conjugated system , sulfide , enzyme , biochemistry , organic chemistry , polymer , computer science , oceanography , programming language , geology , operating system
Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n ‐octanoyl chain on a lipoyl carrier protein (LCP). LipA contains two [4Fe‐4S] clusters, one of which is required to reductively cleave S ‐adenosylmethionine to generate two 5′‐deoxyadenosyl 5′‐radical intermediates that abstract hydrogen atoms from C6 and C8 of the octanoyl chain, while the second cluster has been hypothesized to donate the inserted sulfur atoms. Thus, a single turnover inactivates LipA due to destruction of its second [4Fe‐4S] cluster. Herein, we provide compelling evidence for this controversial role for a [4Fe‐4S] cluster in enzymatic catalysis. Reactions conducted under conditions wherein only one sulfur atom is inserted into the organic substrate results in the LCP substrate becoming cross‐linked to LipA through the Fe/S cluster. Extensive characterization of the cross‐linked species by analytical techniques, Mössbauer spectroscopy, and X‐ray crystallography shows that it contains 3 iron and 3 sulfide ions in a cubane‐like structure that is connected to the LCP by a bridging monothiolated octanoyl linker. HYSCORE spectroscopy was used to demonstrate a direct interaction between a substrate radical, generated using a conjugated substrate analogue, and an iron atom of an Fe/S cluster of LipA.