The Iron-Sulfur Cluster of Pyruvate Formate-Lyase Activating Enzyme in Whole Cells: Cluster Interconversion and a Valence-Localized [4Fe-4S](2+) State

• Type of Material: Article
• Authors: Jian Yang, Michigan State University; Sunil Naik, Emory University; Danilo O. Ortillo, Emory University; Ricardo Garcia-Serres, Emory University; Meng Li, Michigan State University; William E. Broderick, Montana State University; B H Vincent Huynh, Emory University; Joan B. Broderick, Montana State University
• Language: English
• Date: 2009-10-06
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2009 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/bi9010286
• Title of Journal or Parent Work: Biochemistry
• ISSN: 0006-2960
• Volume: 48
• Issue: 39
• Start Page: 9234
• End Page: 9241
• Grant/Funding Information: This work was supported by grants from the National Institutes of Health (GM54608 to J.B.B. and GM47295 to B.H.H.)
• Keywords: PROTEIN SUPERFAMILY; EXPRESSION; ESCHERICHIA-COLI; 4FE-4S; MOSSBAUER-SPECTROSCOPY; SITE; BIOTIN SYNTHASE; Science & Technology; S-ADENOSYLMETHIONINE; GENE; Life Sciences & Biomedicine; RADICAL-SAM; Biochemistry & Molecular Biology
• Research Categories: Chemistry, Biochemistry

Abstract

Pyruvate formate-lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on pyruvate formate-lyase (PFL). Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S]+ cluster state being catalytically active. Such cluster interconversions could play a role in regulating the activity of PFL-AE, and thus of PFL, in response to oxygen levels in vivo. Here we report a Mössbauer investigation on whole cells overexpressing PFL-AE following incubation under aerobic and/or anaerobic conditions and provide evidence that PFL-AE undergoes cluster interconversions in vivo. After 2 h aerobic induction of PFL-AE expression, approximately 44% of the total iron is present in [4Fe-4S]2+ clusters, 6% in [2Fe-2S] 2+ clusters, and the remainder as noncluster FeIII (29%) and FeII (21%) species. Subsequent anaerobic incubation of the culture results in approximately 75% of the total iron being present as [4Fe-4S]2+ clusters, with no detectable [2Fe-2S]2+. Ensuing aerobic incubation of the culture converts the iron species nearly back to the original composition (42% [4Fe-4S]2+, 10% [2Fe-2S] 2+, 19% FeIII, and 29% FeII). The results provide evidence for changes in cluster composition of PFL-AE in response to the redox state of the cell. Furthermore, the Mössbauer spectra reveal that the [4Fe-4S]2+ cluster of PFL-AE in whole cells contains a valence-localized FeIIIFeII pair which has not been previously observed in the purified enzyme. Addition of certain small molecules containing adenosyl moieties, including 5′-deoxyadenosine, AMP, ADP, and methylthioadenosine, to purified PFL-AE reproduces the valence-localized state of the [4Fe-4S]2+ cluster. It is speculated that the [4Fe-4S] 2+ cluster of PFL-AE in whole cells may be coordinated by a small molecule, probably AMP, and that such coordination may protect this labile cluster from oxidative damage.