RESUMO
PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.
Assuntos
Proteínas de Bactérias/química , Citocromos a/química , Geobacter/química , Proteínas Mutantes/química , Proteínas de Bactérias/genética , Cristalografia por Raios X/métodos , Citocromos a/genética , Bases de Dados de Proteínas , Escherichia coli/química , Escherichia coli/genética , Geobacter/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Mutação , Periplasma/química , Periplasma/genética , Conformação Proteica , Proteômica/métodos , Difração de Raios XRESUMO
Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/isolamento & purificação , Citocromos a3/genética , Citocromos a3/isolamento & purificação , Citocromos a/genética , Citocromos a/isolamento & purificação , Citocromos c/química , Escherichia coli/genética , Subunidades Proteicas/química , Clonagem Molecular , Grupo dos Citocromos c/biossíntese , Grupo dos Citocromos c/química , Citocromos a/biossíntese , Citocromos a/química , Citocromos a3/biossíntese , Citocromos a3/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa(3), which is a quinol oxidase, and cytochrome caa(3), which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, Cu(A). B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa(3) but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa(3). B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the Cu(A) centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.
