Transmutation of a heme protein

Area of Science:

• Biochemistry
• Molecular Biology
• Spectroscopy

Background:

• Cytochrome b5 is a key hemoprotein involved in various metabolic processes.
• Understanding the structure-function relationship of cytochrome b5 is crucial for elucidating its biological roles.
• Site-directed mutagenesis provides a powerful tool to investigate protein structure and function.

Purpose of the Study:

• To investigate the structural and functional consequences of replacing Asn57 with Cys in bovine liver cytochrome b5.
• To characterize the different forms of the cytochrome b5 variant generated in recombinant Escherichia coli.
• To propose a mechanism for the formation of these derivatives and discuss their implications for heme and chlorin biosynthesis.

Main Methods:

• Site-directed mutagenesis to create the Asn57Cys variant.
• Recombinant expression in Escherichia coli.
• Characterization using electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry.

Main Results:

• Isolation of four major species (A, BI, BII, C) of the cytochrome b5 variant.
• Identification of form A with a heme group covalently bound via a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group.
• Characterization of forms BI and BII with thioether linkages involving the beta carbon of the heme 4-vinyl group, with BII having an oxidized sulfur.
• Identification of form C as a green species with a noncovalently bound chlorin prosthetic group.

Conclusions:

• The Asn57Cys mutation leads to the formation of diverse cytochrome b5 derivatives with covalently and non-covalently bound prosthetic groups.
• A mechanism for the generation of these derivatives involving thioether bond formation and potential chlorin formation is proposed.
• These findings offer insights into the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups.