Cytochrome rC₅₅₂, Formed during Expression of the Truncated, Thermus thermophilus Cytochrome c₅₅₂ Gene in the Cytoplasm of Escherichia coli, Reacts Spontaneously To Form Protein-Bound 2-Formyl-4-vinyl (Spirographis) Heme^,

Abstract

Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome c(552) in the cytoplasm of Escherichia coli yields both dimeric (rC(557)) and monomeric (rC(552)) cytochrome c-like proteins [Keightley, J. A., et al. (1998) J. Biol. Chem. 273, 12006-12016], which form spontaneously without the involvement of cytochrome c maturation factors. Cytochrome rC(557) is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276, 6537-6544]. Unexpectedly, the monomeric rC(552) transforms spontaneously to a cytochrome-like chromophore having, in its reduced state, the Q(oo) transition (alpha-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC(552), at 1.41 A resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH(2)-S-CH(2)-C(alpha)] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 A resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Q(oo)() transition, the presence of a new, high-frequency band in the resonance Raman spectra at 1666 cm(-1) for oxidized and at 1646 cm(-1) for reduced samples, and the greatly altered, paramagnetically shifted (1)H NMR spectrum observed for this species. The overall process defines a novel mechanism for oxidation of the 2-vinyl group to a 2-formyl group and adds to the surprising array of chemical reactions that occur in the interaction of heme with the CXXCH sequence motif in apocytochromes c.

During Cytochrome c Maturation CcmI Chaperones the Class I Apocytochromes until the Formation of Their b-Type Cytochrome Intermediates

“…They are also unaffected by the redox state of the Cys residues at the hemebinding sites of apocytochromes. apocytochromes from thermophilic organisms like Hydrogenobacter thermophilus cytochrome c 552 (51), Aquifex aeolicus cytochrome c 555 (60), Thermus thermophilus cytochrome c 552 (61), and H. thermoluteolus cytochrome cЈ (62) exhibit secondary structures with high helical contents in the absence of heme (52,62,63) and readily yield b-type cytochrome derivatives upon heme availability in vitro (64). Even in exceptional cases, thermophilic molten globular c-type apocytochromes yield b-type cytochrome intermediates that are conducive to spontaneous covalent heme ligation (51).…”

The Interaction of Covalently Bound Heme with the Cytochrome c Maturation Protein CcmE

“…In a previous RR study using reconstituted Mb with 2-or 4-formylprotoporphyrin IX, the formyl CϭO stretching mode was observed at 1648 cm Ϫ1 (position 2) and 1660 cm Ϫ1 (position 4) in the ferrous form (45). The x-ray structure of a truncated cytochrome c expressed in E. coli, from which the N-terminal signal sequence was removed, showed that the 2-vinyl group of heme was oxidized by an unknown route to a formyl group (46). On the basis of these previous observations, we cannot exclude the possibility that a formyl group is present on the heme of the Y134F variant.…”

The broad diversity of heme-protein cross-links: An overview

“…The observation is further supported by the crystal structure of cyt rc 552 (PDB ID: 1QYZ, Fig. 2F), where Cys11 forms the unusual Cys-heme linkage with heme 2-vinyl group, likely by a nucleophilic or a free radical mechanism involving O 2 (Scheme 1) [34]. Similar to that for cyt rc 552 , N57C/S71C cyt b 5 was overexpressed in E. coli where the cross-links formed spontaneously without the involvement of Ccm apparatus.…”

“…Moreover, resonance Raman studies revealed that the 2-vinyl rather than 4-vinyl group of heme was covalently linked to CcmE [74]. With regard to the mechanism, the formation of an unusual His-heme cross-link may involve a radical following the anti-Markovnikov rule, as proposed for Cys [34], Met [53] or SeCys [61] cross-linking to the C β atom of vinyl group. As pointed out by the authors [73], this unusual His-heme cross-link has unique features that allow the formation of a transient covalent complex with CcmE for the protein to function as a heme chaperone.…”

“…In the absence of an X-ray or NMR structure, study on the M245T mutant revealed the reduction of heme vinyl group and the Scheme 1. Proposed nucleophilic and free radical mechanism for formation of an unusual [heme-CO-CH 2 -S-CH 2 -C α ] linkage [34].…”