INVOLVEMENT OF PRODUCTS OF THE NRFEFG GENES IN THE COVALENT ATTACHMENT OF HEME-C TO A NOVEL CYSTEINE-LYSINE MOTIF IN THE CYTOCHROME-C(552) NITRITE REDUCTASE FROM ESCHERICHIA-COLI

Cytochrome c(552) is the terminal component of the formate-dependent n itrite reduction pathway of Escherichia coli. In addition to four 'typ ical' haem-binding motifs, CXXCH-, characteristic of c-type cytochrome s, the N-terminal region of NrfA includes a motif, CWSCK, Peptides gen erated by digesting the cytochrome from wild-type bacteria with cyanog en bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to ha em, was generated by fragmentation of a peptide of mass 1312 that incl uded the sequence CWSCK, Neither this signal nor the haem-containing p eptide of mass 1312 was detected in parallel experiments with cytochro me that had been purified from a transformant unable to synthesize Nrf E, NrfF and NrfG: this is consistent with our previous report that Nrf E and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low midpoint potential redox centres. The best fit to the experimental data is for three n=1 components with midpoint redox potentials (pH 7 .0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210 mV (43% of the total). Plasmids in which the lysine c odon of the cysteine-lysine motif, AAA, was changed to the histidine c odon 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 nit rite reduction to the transformants. The presence of a 50 kDa periplas mic 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, alt hough still detectable, were far lower than that of the native protein . The combined data establish not only that there is a haem group boun d covalently to the cysteine-lysine motif of cytochrome c(552) but als o that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.