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

Abstract

SummaryCytochrome c 552 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 haemcontaining 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 midpoint potential redox centres. The best fit to the experimental data is for three n ¼ 1 components with midpoint redox potentials (pH 7.0) of þ45 mV (21% of the total absorbance change), ¹90 mV (36% of the total) and ¹210 mV (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 ¹ deletion mutant to Nrf þ 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 c 552 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.

CytochromecBiogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

“…The apocytochrome c recognition motif for system I has been much studied and appears to be only the CXXCH motif (6-8, 13, 14, 29), with the caveat that CXXXCH may also be used (130). Some variants of system I (e.g., NrfEFG [51], which is related to CcmFGH) and system II (CcsBA) recognize slightly altered apocytochrome c signature motifs such as CXXCK (51,74,117) or CX15CH (73). These variants are likely to play major roles in the future in understanding of the apocytochrome c recognition determinants and the features of the substrate and synthetases that are important for such.…”

Dispensable residues in the active site of the cytochrome c biogenesis protein CcmH

“…The proteins NrfEFG are paralogues of CcmFH, and are involved in the specific heme attachment to an unusual cytochrome motif (CXXCK) in the nitrite reductase NrfA [2,9]. The NrfG structure adds weight to the previous suggestion that the TPR motifs observed in the sequences of C-CcmH/CcmI/NrfG are involved in interaction with the cytochrome substrate.…”

Cytochrome c assembly

“…CcmF is regarded as the ''heme lyase'' protein of the Ccm system (56), although no mechanism has been proposed. A paralogue of CcmF (NrfE) is required for heme attachment to a cytochrome c (NrfA) containing a CXXCK motif (57). How the System II proteins catalyze thioether bond formation between heme and the apocytochrome cysteines is also not known.…”