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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^†^,^‡

Supporting Info

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, and Andrzej Pastuszyn

Department of Biology, University of California at San Diego, La Jolla, California 92093, Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, Department of Chemistry, Occidental College, Los Angeles, California 90041, Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006-8921, Department of Chemistry/Biochemistry, Eastern Washington University, Cheney, Washington 99004, and Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, New Mexico 87131-5221

Biochemistry, 2004, 43 (38), pp 12162–12176

DOI: 10.1021/bi048968l

Publication Date (Web): September 2, 2004

†

Supported by NIH Grants GM35342 (J.A.F.), GM18865 (T.M.L.), GM63170 (K.L.B.), and GM48495 (C.D.S.).

‡

Structure coordinates of cytochromes rC₅₅₂ and p572 were deposited with the Protein Data Bank as entries 1QYZ and 1ROQ, respectively.

To whom correspondence should be addressed. Telephone: (858) 784-9235. Fax: (858) 784-2857. E-mail: jafee@scripps.edu.

University of California at San Diego.

The Scripps Research Institute.

University of Rochester.

Occidental College.

Oregon Health and Science University.

Eastern Washington University.

Present address: Astex Technology, 436 Cambridge Science Park, Cambridge CB4 OQA, United Kingdom.

Present address: ActiveSight, 4045 Sorrento Valley Blvd., San Diego, CA 92121.

University of New Mexico.

Abstract

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 Q_oo 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-C_α] 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 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 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.