American Chemical Society
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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,

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journal contribution
posted on 2004-09-28, 00:00 authored by James A. Fee, Thomas R. Todaro, Eugene Luna, Donita Sanders, Laura M. Hunsicker-Wang, Kirti M. Patel, Kara L. Bren, Ester Gomez-Moran, Michael G. Hill, Jingyuan Ai, Thomas M. Loehr, W. Anthony Oertling, Pamela A. Williams, C. David Stout, Duncan McRee, Andrzej Pastuszyn
Expression of the truncated (lacking an N-terminal signal sequence) structural gene of Thermus thermophilus cytochrome c552 in the cytoplasm of Escherichia coli yields both dimeric (rC557) and monomeric (rC552) 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 rC557 is comprised of a dimer and has been structurally characterized [McRee, D., et al. (2001) J. Biol. Chem. 276, 6537−6544]. Unexpectedly, the monomeric rC552 transforms spontaneously to a cytochrome-like chromophore having, in its reduced state, the Qoo transition (α-band) at 572 nm (therefore called p572). The X-ray crystallographic structure of rC552, at 1.41 Å resolution, shows that the 2-vinyl group of heme ring I is converted to a [heme-CO-CH2-S-CH2-Cα] conjugate with cysteine 11. Electron density maps obtained from isomorphous crystals of p572 at 1.61 Å resolution reveal that the 2-vinyl group has been oxidized to a formyl group. This explains the lower energy of the Qoo 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 1H 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.