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The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state.

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.

SUBMITTER: Yang J

PROVIDER: S-EPMC4230446 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state.

Yang Jian J 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

Biochemistry 20091001 39

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 Mo ...[more]

PMID: 19711960

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Project description:Iron-sulfur (Fe-S) clusters are ubiquitous and versatile inorganic cofactors that are crucial for many fundamental bioprocesses in nearly all organisms. How cells maintain Fe-S cluster homeostasis is not well understood in Gram-positive bacteria. Genomic analysis showed that the Suf system, which is encoded by the sufRBDCSU operon, is the sole Fe-S cluster assembly system in the genus StreptomycesStreptomyces avermitilis is the industrial producer of avermectins, which are widely used as agricultural pesticides and antiparasitic agents. sufR (SAV6324) encodes a putative ArsR-family transcriptional regulator, which was characterized as a repressor of the sufRBDCSU operon in this investigation. Spectroscopy and mass spectrometry demonstrated that anaerobically isolated SufR contained an oxidation-sensitive [4Fe-4S] cluster and existed as a homodimer. Electrophoretic mobility shift assays (EMSAs) and DNase I footprinting analyses revealed that [4Fe-4S]-SufR bound specifically and tightly to a 14-bp palindromic sequence (CAAC-N6-GTTG) in the promoter region of the sufR operon, repressing expression of the sufRBDCSU operon. The presence of the [4Fe-4S] cluster is critical for the DNA-binding activity of SufR. Cys182, Cys195, and Cys223 in the C-terminal region of SufR are essential for [4Fe-4S] cluster coordination, but Cys178 is not. The fourth non-Cys ligand in coordination of the [4Fe-4S] cluster for SufR remains to be identified. The findings clarify the transcriptional control of the suf operon by [4Fe-4S] SufR to satisfy the various Fe-S cluster demands. SufR senses the intracellular Fe-S cluster status and modulates the expression of the sole Fe-S cluster assembly system via its Fe-S cluster occupancy.IMPORTANCE Fe-S clusters function as cofactors of proteins controlling diverse biological processes, such as respiration, photosynthesis, nitrogen fixation, DNA replication, and gene regulation. The mechanism of how Actinobacteria regulate the expression of the sole Fe-S cluster assembly system in response to the various Fe-S cluster demands remains to be elucidated. In this study, we showed that SufR functions as a transcriptional repressor of the sole Fe-S cluster assembly system in the avermectin producer S. avermitilis [4Fe-4S]-SufR binds to the promoter region of the suf operon and represses its expression. When Fe-S cluster levels are insufficient, SufR loses its [4Fe-4S] cluster and DNA-binding activity. Apo-SufR dissociates from the promoter region of suf operon, and the expression of the suf system is strongly increased by derepression to promote the synthesis of Fe-S clusters. The study clarifies how Streptomyces maintains its Fe-S cluster homeostasis through the activity of SufR to modulate the various Fe-S cluster demands.

Project description:The all-ferrous, carbene-capped Fe(4)S(4) cluster, synthesized by Deng and Holm (DH complex), has been studied with density functional theory (DFT). The geometry of the complex was optimized for several electronic configurations. The lowest energy was obtained for the broken-symmetry (BS) configuration derived from the ferromagnetic state by reversing the spin projection of one of the high spin (S(i) = 2) irons. The optimized geometry of the latter configuration contains one unique and three equivalent iron sites, which are both structurally and electronically clearly distinguishable. For example, a distinctive feature of the unique iron site is the diagonal Fe···S distance, which is 0.3 Å longer than for the equivalent irons. The calculated (57)Fe hyperfine parameters show the same 1:3 pattern as observed in the Mössbauer spectra and are in good agreement with experiment. BS analysis of the exchange interactions in the optimized geometry for the 1:3, M(S) = 4, BS configuration confirms the prediction of an earlier study that the unique site is coupled to the three equivalent ones by strong antiferromagnetic exchange (J > 0 in J ?(j<4)?(4)·?(j)) and that the latter are mutually coupled by ferromagnetic exchange (J' < 0 in J' ?(i<j<4)?(i)·?(j)). In combination, these exchange couplings stabilize an S = 4 ground state in which the composite spin of the three equivalent sites (S(123) = 6) is antiparallel to the spin (S(4) = 2) of the unique site. Thus, DFT analysis supports the idea that the unprecedented high value of the spin of the DH complex and, by analogy, of the all-ferrous cluster of the Fe-protein of nitrogenase, results from a remarkably strong dependence of the exchange interactions on cluster core geometry. The structure dependence of the exchange-coupling constants in the Fe(II)-(?(3)-S)(2)-Fe(II) moieties of the all-ferrous clusters is compared with the magneto-structural correlations observed in the data for dinuclear copper complexes. Finally, we discuss two all-ferric clusters in the light of the results for the all-ferrous cluster.

Dataset Information

The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state.

Publications

The iron-sulfur cluster of pyruvate formate-lyase activating enzyme in whole cells: cluster interconversion and a valence-localized [4Fe-4S]2+ state.

Similar Datasets

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