Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli.
Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid-point potential redox centres. The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.
Eaves
D J
DJ
School of Biochemistry, University of Birmingham, UK.
Grove
J
J
Staudenmann
W
W
James
P
P
Poole
R K
RK
White
S A
SA
Griffiths
I
I
Cole
J A
JA
D000693:Anaerobiosis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D017353:Gene Deletion; D005798:Genes, Bacterial; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009876:Operon; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics
D003574:Cytochrome c Group; C083951:cytochrome c553; D006418:Heme; C022302:cytochrome C-552; D009572:Nitrite Reductases; D008239:Lysine; D003545:Cysteine