Transmutation of a Heme Protein

Residue Asn57of bovine liver cytochrome b5has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy,1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (Em= -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57and the α carbon of the heme 4-vinyl group. Form BIhas a covalently bound heme group coupled through a thioether linkage involving the β carbon of the heme 4-vinyl group. Form BIIis similar to BIexcept that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (Em= 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.