TRANSMUTATION OF A HEME PROTEIN

Residue Asn57 of bovine liver cytochrome b5 has been replaced with a c ysteine residue, and the resulting variant has been isolated from reco mbinant Escherichia coli as a mixture of four major species: A, B(I), B(II), and C. A combination of electronic spectroscopy, H-1 NMR spectr oscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four m ajor cytochrome derivatives. The red form A (E(m) = -19 mV) is found t o possess a heme group bound covalently through a thioether linkage in volving Cys57 and the alpha carbon of the heme 4-vinyl group. Form B(I ) has a covalently bound heme group coupled through a thioether linkag e involving the beta carbon of the heme 4-vinyl group. Form B(II) is s imilar to B(I) except that the sulfur involved in the thioether linkag e is oxidized to a sulfoxide. The green form C (E(m) = 175 mV) possess es a noncovalently bound prosthetic group with spectroscopic propertie s characteristic of a chlorin. A mechanism is proposed for the generat ion of these derivatives, and the implications of these observations f or the biosynthesis of cytochrome c and naturally occurring chlorin pr osthetic groups are discussed.