Gene Synthesis, Bacterial Expression, and 1H NMR Spectroscopic Studies of the Rat Outer Mitochondrial Membrane Cytochrome B5

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The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome bs (OM cytochrome b5) from rat liver has been synthesized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102], The recombinant OM cytochrome b5was characterized by UV-visible, EPR, and 1HNMR spectroscopy. The UV-visible and EPR spectra of the OMcytochrome bs are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome bs [Bodman, S.B.V., Schyler, M.A., Jollie, D.R., & Sligar, S.G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional NMR spectrum of the OM cytochrome bs indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes bs. Two-dimensional NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its α-γ-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 ± 0.1. It is noteworthy that the two cytochromes bs that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver. © 1992, American Chemical Society. All rights reserved.

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