The roles of different regions of the CycH protein in c-type cytochrome biogenesis in Sinorhizobium meliloti.
Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c. The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery. We propose that these two modules of CycH play roles in different functions of the protein. The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis. Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein. TPR domains are known to mediate protein-protein interactions. Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr-) and nitrogen-fixing (Fix-) phenotypes. We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr+ phenotype. In contrast, only the full-length protein confers the ability to fix nitrogen.
Cinege
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G
Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, PO Box 521, 6701 Szeged, Hungary. cinege@nucleus.szbk.u-szeged.hu
Kereszt
A
A
Kertész
S
S
Balogh
G
G
Dusha
I
I
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D016962:Sinorhizobium meliloti / Q000235:genetics*; D001616:beta-Galactosidase
D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D006418:Heme; D045304:Cytochromes c; D001616:beta-Galactosidase