PMID:19711960
| Citation |
Yang, J, Naik, SG, Ortillo, DO, García-Serres, R, Li, M, Broderick, WE, Huynh, BH and Broderick, JB (2009) The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state. Biochemistry 48:9234-41 |
|---|---|
| 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 Mossbauer 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 Fe(III) (29%) and Fe(II) (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% Fe(III), and 29% Fe(II)). The results provide evidence for changes in cluster composition of PFL-AE in response to the redox state of the cell. Furthermore, the Mossbauer spectra reveal that the [4Fe-4S](2+) cluster of PFL-AE in whole cells contains a valence-localized Fe(III)Fe(II) 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. |
| Links |
PubMed Online version:10.1021/bi9010286 |
| Keywords |
Electrons; Enzyme Activation/physiology; Enzymes/chemistry; Enzymes/genetics; Enzymes/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Ferric Compounds/chemistry; Ferric Compounds/metabolism; Ferrous Compounds/chemistry; Ferrous Compounds/metabolism; Gene Expression Regulation, Bacterial; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/genetics; Iron-Sulfur Proteins/metabolism; Spectroscopy, Mossbauer |
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