The histidine of the c-type cytochrome CXXCH haem-binding motif is essential for haem attachment by the Escherichia coli cytochrome c maturation (Ccm) apparatus

Abstract

c-type cytochromes are characterized by covalent attachment of haem to the protein by two thioether bonds formed between the haem vinyl groups and the cysteine sulphurs in a CXXCH peptide motif. In Escherichia coli and many other Gram-negative bacteria, this post-translational haem attachment is catalysed by the Ccm (cytochrome c maturation) system. The features of the apocytochrome substrate required and recognized by the Ccm apparatus are uncertain. In the present study, we report investigations of maturation of cytochrome b562 variants containing CXXCR, CXXCK or CXXCM haem-binding motifs. None of them showed any evidence for correct maturation by the Ccm system. However, we have determined, for each variant, that the proteins (i) were expressed in large amounts, (ii) could bind haem in vivo and/or in vitro and (iii) were not degraded in the cell. Together with previous observations, these results strongly suggest that the apocytochrome substrate feature recognized by the Ccm system is simply the two cysteine residues and the histidine of the CXXCH haem-binding motif. Using the same experimental approach, we have also investigated a cytochrome b562 variant containing the special CWSCK motif that binds the active-site haem of E. coli nitrite reductase NrfA. Whereas a CWSCH analogue was matured by the Ccm apparatus in large amounts, the CWSCK form was not detectably matured either by the Ccm system or by the dedicated Nrf biogenesis proteins, implying that the substrate recognition features for haem attachment in NrfA may be more extensive than the CWSCK motif.

Figure 1. Absorption spectra of c-type cytochrome variants of E. coli cytochrome b₅₆₂

(A) Unpurified periplasmic extracts of E. coli transformed with plasmids for the E. coli Ccm system and cytochrome b₅₆₂ with a CNACH variant haem-binding motif (after Allen et al. [7]). The spectrum was recorded at 25 °C after the addition of a few grains of dithionite to the periplasmic proteins in 100 mM Tris/HCl (pH 8.0), 0.5 mM EDTA and 0.25 M sucrose. The spectral maxima were 420, 526 and 556 nm. On the basis of the data and molar absorption coefficients given in [6,7], the holocytochrome concentration was 5.8 μM. Such a holocytochrome when purified was independently examined by NMR spectroscopy and found to be properly matured, i.e. homogeneous, with haem covalently attached to the protein through two thioether bonds with the ‘correct’, universally conserved, stereochemistry [7]. (B) E. coli holocytochrome b₅₆₂ with a CXXCM variant haem-binding motif. This protein was purified from periplasmic extracts of E. coli using Q-Sepharose chromatography resin and was subsequently reduced with disodium dithionite. The spectrum was recorded at 25 °C in 50 mM Tris/HCl (pH 8.0). Observed spectral maxima were 429, 526 and 557 nm, with a prominent shoulder at approx. 422 nm. There is no molar absorption coefficient available for this protein, but based on the data in [6], the holocytochrome concentration can be estimated as approx. 1.2 μM. (C) Unpurified periplasmic extracts of E. coli transformed with the plasmid for E. coli cytochrome b₅₆₂ with a CNACK variant haem-binding motif. The spectrum was recorded at 25 °C after the addition of a few grains of dithionite to the periplasmic proteins in 100 mM Tris/HCl (pH 8.0), 0.5 mM EDTA and 0.25 M sucrose. The spectral maxima were 423, 529 and 559 nm. There is no molar absorption coefficient available for this protein, but based on the data in [6], the holocytochrome concentration can be estimated as approx. 3.3 μM. (D) Unpurified periplasmic extracts of E. coli transformed with plasmids for the E. coli Ccm system and cytochrome b₅₆₂ with a CWSCH variant haem-binding motif. The spectrum was recorded at 25 °C after the addition of a few grains of dithionite to the periplasmic proteins in 20 mM Tris/HCl (pH 8.0), 0.1 mM EDTA and 0.1 M sucrose. The spectral maxima were 420, 526 and 556 nm. On the basis of the data and molar absorption coefficients given in [6,7], the holocytochrome concentration was 5.5 μM. Inset: reduced pyridine haemochrome spectrum of the same periplasmic extract. Final concentrations of hydroxide and pyridine were 0.2 M and 30% (v/v) respectively. The peak maximum was at 550.0 nm, characteristic of haem attachment to the protein through two thioether bonds.