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Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

ABSTRACT: Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.

SUBMITTER: Ruprecht J

PROVIDER: S-EPMC3069475 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

Ruprecht Jonathan J Iwata So S Rothery Richard A RA Weiner Joel H JH Maklashina Elena E Cecchini Gary G

The Journal of biological chemistry 20110210 14

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the re ...[more]

PMID: 21310949

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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.

Dataset Information

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

Publications

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

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