Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.
NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the cleavage site showed it was between two equally sized di-haem domains predicted from sequence analysis.
Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.
Michaël L
ML
Cartron
M Dolores
MD
Roldán
Stuart J
SJ
Ferguson
Ben C
BC
Berks
David J
DJ
Richardson
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016227:Benzoquinones / Q000378:metabolism; D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006418:Heme / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010230:Paracoccus / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D033903:Periplasmic Proteins / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D016227:Benzoquinones; D003574:Cytochrome c Group; D006873:Hydroquinones; D008024:Ligands; D033903:Periplasmic Proteins; C004532:quinone; D006418:Heme; D006639:Histidine; C017165:cytochrome c(3); D007501:Iron; D010088:Oxidoreductases; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C077913:periplasmic protein disulfide oxidoreductase