Biology Education

A suggested MSc student project in bioinformatics potentially comprising also biochemistry and molecular microbiology studies.

Supervisors: Dr. Courtney Stairs and professor emeritus Lars Hederstedt

Background

Succinate:quinone oxidoreductase (SQR) is both a Krebs´ citric acid cycle enzyme and a component (Complex II) of the respiratory chain in aerobic cells. The enzyme has a central role in carbon and energy metabolism by catalyzing the oxidation of succinate to form fumarate coupled to the reduction of quinone in the respiratory chain. SQR consists of a membrane-peripheral heterodimer domain (often called succinate dehydrogenase) which is tightly bound to a membrane-spanning anchor, which is a cytochrome with one or two heme groups depending on the organism. The peripheral domain is composed of a flavoprotein subunit (Fp or SdhA), with a covalently bound FAD group, and an iron-sulfur protein subunit (Ip or SdhB) with three iron-sulfur clusters (one each of a [2Fe-2S], a [3Fe-4S] and a [4Fe-4S cluster]). The flavin together with the iron-sulfur clusters function to mediate electron transfer from the dicarboxylate binding site on the Fp subunit to the membrane-anchor domain where quinone is reduced to quinol. The Fp and Ip subunits are both very conserved with respect to composition and polypeptide sequence [1]. The membrane anchor of SQR in mammalian mitochondria and proteobacteria, such as Escherichia coli, consists of two polypeptides (SdhC and SdhD) and contains one heme group. In Bacillus subtilis, the anchor is a single polypeptide (SdhC) containing two heme groups [2]. The Fp and Ip subunits are post-translationally processed in the cytoplasm. With the full complement of prosthetic groups they are then bound to the heme-containing membrane-anchor to form the active SQR. If the membrane-anchor is missing or defective, Fp and Ip subunits accumulate as soluble proteins in the cytoplasm. Flavinylation of Fp in B. subtilis does not require the Ip subunit or the membrane anchor but depends on folding of the Fp polypeptide into a state that can bind FAD and subsequent mediate covalent binding of the FAD [3, 4].

About 10% of all different flavoproteins contain covalently attached flavin via linkage to the isoalloxazine ring [5]. In the Fp subunit of SQR, and the closely related membrane-associated fumarate reductase of anaerobic organisms, FAD is covalently attached via an 8α-N(3)-histidyl linkage to a histidine (His) in a conserved sequence in the N-terminal part of the polypeptide. The covalent bond to FAD in SQR is essential for enzyme activity and the main reason for this is that the bond raises the midpoint redox potential of the flavin by more than 80 mV compared to non-covalently bound flavin. SQR in E. coli and B. subtilis can be assembled in the membrane without covalently bound FAD but are enzymatically inactive [3, 4, 6].

In 1987 the sdhCAB operon of B. subtilis was cloned and sequenced. The operon on plasmid in E. coli was found to result in the production of all three subunit polypeptides [7]. However, the E. coli cells did not show increased succinate dehydrogenase activity nor did the operon complement a sdhCDAB defective E. coli mutant [7]. The B. subtilis Fp polypeptide produced in E. coli was not membrane bound and migrated slightly differently upon SDS-polyacrylamide gel electrophoresis compared to that produced in B. subtilis. Notably, the N-terminal of the Fp polypeptide was processed identically in the two bacteria [7, 8]. The B. subtilis Fp polypeptide was found to lack covalently bound FAD when produced in E. coli although E. coli Fp in the same cell was flavinylated [8]. From these findings it was at the time concluded that B. subtilis Fp is not flavinylated in E. coli because one or more host-specific factors are required or flavinylation is somehow prevented. It is unexpected that B. subtilis SQR without covalently bound FAD is not assembled in the E. coli membrane provided that all other aspects of assembly, including iron-sulfur cluster biogenesis, are functional in the heterologous system. Notably, certain B. subtilis mutants with mutated Fp lack covalently bound flavin and do not assemble the SQR polypeptides in the membrane [3]. The reason for the observed defective assembly in E. coli is probably not some unidentified mutation in the plasmid DNA because the same plasmid preparation complemented a B. subtilis sdhCAB deletion strain [7]. The slight difference in electrophoretic mobility of normal Fp and that produced in E. coli could be due to the absence of covalently FAD but could have another explanation because this difference was not observed for some mutant Fp without covalently bound FAD [3].

Now, more than 25 years later, SdhE in E. coli and SDHAF2 in mammalian cells, respectively, has been identified as an assembly factor important for the covalent binding of FAD to the Fp polypeptide of SQR. SdhE and SDHAF2 are homologous proteins. SdhE is not important for the binding of FAD to folded SdhA but promotes the autocatalytic covalent binding to the protein. As far as known to date [9], SdhE and its homologs bind to the folded Fp polypeptide close to the dicarboxylate binding site and in the region where Ip binds in the final assembled SQR. SdhE is remarkably not essential for the formation of the covalent bond, i.e., the bond forms efficiently in vitro using isolated apo-Fp, sufficient concentrations of FAD, and certain buffer conditions. Inclusion of succinate or some other carboxylic acid in the buffer promotes formation of the covalent bond. The function of SdhE is thus as a chaperone increasing the rate of covalent bond formation and reducing the concentration of FAD needed for the reaction to proceed efficiently. The formation of the covalent bond is autocatalytic based on radical chemistry initiated via the FAD when half-reduced [9].

Many bacteria, including Bacillus species, apparently lack SdhE and it remains an open question if these organisms do not require a protein factor for the covalent binding of FAD to Fp. There is no indication from genetic data for a specific assembly factor involved in SQR assembly in B. subtilis. All mutations (>30) affecting succinate dehydrogenase activity identified so far in B. subtilis map within the sdhCAB operon [10]. Thus, if there is a specific protein required for the flavinylation of the Fp subunit, the gene for this protein is probably essential for cell growth or there are perhaps two proteins in the cell with overlapping activity.

In summary, the requirements for covalent binding of FAD to the SdhA polypeptide in B. subtilis is not known and the reason to why B. subtilis SdhA does not bind FAD covalently when produced in E. coli remains elusive.

Research questions

1. Is there in Firmicute bacteria any protein similar to SdhE/SDHAF2? Compare with published data on the distribution of SdhE in bacteria [11].
2. Is there in Firmicute bacteria any protein-coding gene that is frequently associated with sdhA but of unknown function? The sdhE gene in coli and Paracoccus denitrificans is found in the vicinity of sdhA on the chromosome.
3. Does subtilis SdhA contain non-covalently bound FAD when produced in E. coli? If not, it would explain the lack of covalently bound FAD. For the experiments one could make use of a recombinant inducible gene on plasmid encoding His₆-tagged B. subtilis SdhA. The tagged protein would be produced in E. coli, isolated by affinity chromatography and finally analyzed for flavin content and electrophoretic migration (detection by protein stain and/or Western blot). Depending on results the resulting polypeptide with wild type Fp as reference could by analyzed by mass spectrometry
4. Irrespective of the result under C, purified SdhA can be used to investigate whether covalently bound FAD can be formed in vitro, just as has been done [9] with E. coli SdhA.
5. Is subtilis Fp flavinylated in E. coli if SdhE and/or SdhB is missing? This is to test whether possibly SdhE or B. subtilis SdhB interferes with flavinylation.
6. One mutation in subtilis sdhA that causes a defective flavinylation of Fp has not yet been identified. The mutation can be identified by DNA sequence analysis of the mutant gene amplified from chromosomal DNA using a pair of specific primers.

References

Key references (essential and suggested for startup reading) are indicated in bold font.

1. Karavaeva, V. & Sousa, F. L. (2023) Molecular structure of complex II: An evolutionary perspective, Biochem Biophys Acta Bioenergetics. 1864.
2. Hederstedt, L. (2002) Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis, Biochim Biophys Acta. 1553, 74-83.
3. Hederstedt, L. (1983) Succinate dehydrogenase mutants of Bacillus subtilis lacking covalently bound flavin in the flavoprotein subunit, Eur J Biochem. 132, 589-593.
4. Maguire, J., Magnusson, K. & Hederstedt, L. (1986) Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: Identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme, Biochemistry. 25, 5202-5208.
5. Heuts, D. P., Scrutton, N. S., McIntire, W. S. & Fraaije, M. W. (2009) What´s in a covalent bond? On the role and formation of covalently bound flavin cofactors, FEBS J. 276, 3405-3427.
6. E., M., Rajagukguk, S., Starbird, C. A., McDonald, W. H., Koganitsky, A., Eisenbach, M. & al, e. (2016) Binding of the covalent flavin assembly factor to the flavoprotein subunit of complex II, J Biol Chem. 291, 2904-2916.
7. Phillips, M. K., Hederstedt, L., Hasnain, S., Rutberg, L. & Guest, J. R. (1987) Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex, J Bacteriol. 169, 864-873.
8. Hederstedt, L., Bergman, T. & Jornvall, H. (1987) Processing of Bacillus subtilis succinate dehydrogenase and cytochrome b-558 polypeptides. Lack of covalently bound flavin in the Bacillus enzyme expressed in Escherichia coli, FEBS Lett. 213, 385-90.
9. Maklashina, E., Iverson, T. M. & Cecchini, G. (2022) How an assembly factor enhances covalent FAD attachment to the flavoprotein subunit- of complex II, J Biol Chem. 298, 102472.
10. Hederstedt, L., Magnusson, K. & Rutberg, L. (1982) Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion, J Bacteriol. 152, 157-65.
11. McNeil, M. B., Clulow, J. S., Wilf, N. M., Salmond, G. P. & Fineran, P. C. (2012) SdhE is a conserved protein required for flavinylation of succinate dehydrogenase in bacteria, J Biol Chem. 287, 18418-18428.

January 7, 2024,

Lars Hederstedt