The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state.
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.
Yang
Jian
J
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Naik
Sunil G
SG
Ortillo
Danilo O
DO
García-Serres
Ricardo
R
Li
Meng
M
Broderick
William E
WE
Huynh
Boi Hanh
BH
Broderick
Joan B
JB
D000123:Acetyltransferases; D004583:Electrons; D004789:Enzyme Activation / Q000502:physiology; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015204:Spectroscopy, Mossbauer
D004798:Enzymes; D029968:Escherichia coli Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D007506:Iron-Sulfur Proteins; D000123:Acetyltransferases; C040285:pyruvate formate-lyase activating enzyme