| 1. |
- Fridén, H, et al.
(författare)
-
Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558
- 1987
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 168, s. 695-701
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.
|
|
| 2. |
- Hansson, Mats, et al.
(författare)
-
Purification and characterisation of a water-soluble ferrochelatase from Bacillus subtilis
- 1994
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 220:1, s. 201-208
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus subtilis ferrochelatase is encoded by the hemH gene of the hemEHY gene cluster and catalyses the incorporation of Fe2+ into protoporphyrin IX. B. subtilis ferrochelatase produced in Escherichia coli was purified. It was found to be a monomeric, water-soluble enzyme of molecular mass 35 kDa which in addition to Fe2+ can incorporate Zn2+ and Cu2+ into protoporphyrin IX. Chemical modification experiments indicated that the single cysteine residue in the ferrochelatase is required for enzyme activity although it is not a conserved residue compared to other ferrochelatases. In growing B. subtilis, the ferrochelatase constitutes approximately 0.05% (by mass) of the total cell protein, which corresponds to some 600 ferrochelatase molecules/cell. The turnover number of isolated ferrochelatase, 18-29 min-1, was found to be consistent with the rate of haem synthesis in exponentially growing cells (0.2 mol haem formed/min/mol enzyme). It is concluded that the B. subtilis ferrochelatase has enzymic properties which are similar to those of other characterised ferrochelatases of known primary structure, i.e. ferrochelatases of the mitochondrial inner membrane of yeast and mammalian cells. However, in contrast to these enzymes the B. subtilis enzyme is a water-soluble protein and should be more amenable to structural analysis.
|
|
| 3. |
- Svensson, Birgitta, et al.
(författare)
-
Low spin heme A in the heme A biosynthetic protein CtaA from Bacillus subtilis
- 1996
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; , s. 287-295
-
Tidskriftsartikel (refereegranskat)abstract
- Synthesis of heme A from heme B (protoheme IX) most likely occurs in two steps with heme O as an intermediate. Bacillus subtilis CtaB, an integral membrane protein, functions in farnesylation of heme B to form heme O. CtaA, also a membrane protein, is required for heme A synthesis from heme O and appears to be a monooxygenase and/or a dehydrogenase. Wild-type ctaA and ctaB expressed together from plasmids in B. subtilis resulted in CtaA containing equimolar amounts of low-spin heme B and heme A; this form of CtaA was named cyt ba -CTA. A mutant ctaB gene was identified and characterised. It encodes a truncated CtaB polypeptide. Wild-type ctaA and the mutant ctaB gene on plasmids resulted in CtaA containing mainly low-spin heme B; this variant was named cyt b -CTA. The heme B component in cyt ba -CTA and cyt b -CTA showed identical properties; a mid-point redox potential of +85 mV, an EPR gmax signal at 3.7, and a split α-band light absorption peak. The heme A component in cyt ba -CTA showed a mid-point potential of +242 mV, an EPR gmax signal at 3.5, and the α-band light absorption peak at 585 nm. It is suggested that the CtaA protein contains two heme binding sites, one for heme B and one for substrate heme. The heme B would play a role in electron transfer, i.e. function as a cytochrome, in the monooxygenase and/or dehydrogenase reaction catalysed by CtaA whereas heme O/heme A would be substrate/product.
|
|
| 4. |
- von Wachenfeldt, Claes, et al.
(författare)
-
Physico-chemical characterisation of membrane-bound and water-soluble forms of Bacillus subtilis cytochrome c-550
- 1993
-
Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033.
-
Tidskriftsartikel (refereegranskat)abstract
- Cytochrome c-550 of the Gram-positive bacterium, Bacillus subtilis, is a membrane-bound 13-kDa protein encoded by the cccA gene. The cytochrome has been proposed to be comprised of an N-terminal membrane anchor domain (about 30 residues) which spans the cytoplasmic membrane in an alpha-helical conformation and a C-terminal heme domain (about 70 residues) which is located on the outside of the cytoplasmic membrane. Cytochrome c-550 was purified in the presence of Triton X-100 and characterised. In the reduced state it shows absorption maxima at 415, 521, 550 nm and in the oxidised state a Soret band at 408 nm and a weak band at about 695 nm. The latter absorption band, together with data from amino acid sequence comparisons, strongly suggest His64 and Met99 as the fifth and sixth axial ligands to the heme iron in cytochrome c-550. The midpoint redox potential of the cytochrome, +178 mV, was pH-independent in the pH range 6.0-7.9. Oxidised cytochrome c-550 showed an EPR signal at g(max) = 3.41, which is unusual for low-spin cytochromes c with His/Met axial ligation. The heme domain was isolated as a tryptic fragment of 74 residues and as a protein-A-cytochrome-c-550 hybrid protein. Both these forms were water-soluble and showed thermodynamic and spectroscopic properties indistinguishable from the membrane-bound form of cytochrome c-550 and are suitable for structural analysis of the heme domain by X-ray crystallography or NMR techniques. Polypeptide analysis of the membrane-bound and water-soluble tryptic fragment confirmed that B. subtilis cytochrome c-550 in the membrane consists of 120 amino acid residues and has a two-domain structure.
|
|
