Gene synthesis, bacterial expression and proton NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5

Abstract

The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized 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 b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [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 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H 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 alpha-gamma-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 b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.

Identification of Crucial Histidines for Heme Binding in the N-terminal Domain of the Heme-regulated eIF2α Kinase

“…A similar UV-visible spectrum was also encountered for ferric cytochrome b 5 , a typical bishistidine-ligated hemoprotein (9) (Table II). While the resonance Raman spectrum of the ferric NTD exhibited a weak 3 line at 1473 cm Ϫ1 , implying the presence of a five-coordinate heme species, an intense 3 line characteristic of a six-coordinate heme species was observed at 1506 cm Ϫ1 (Table III).…”

Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17

“…Curve fitting and calculations of maximum velocity (V max ) and apparent Michaelis constant (K m ) values were performed using LEONORA (48). (51)(52)(53). To identify the forms of cytochrome b 5 found in NCI-H295A cells, we performed RT-PCR with two pairs of oligonucleotide primers that will amplify the three products of the two genes.…”

Molecular Cloning, Overexpression in Escherichia coli, Structural and Functional Characterization of House Fly Cytochrome b5

“…Because of the unpaired electron of the low spin Fe(III) heme center, many of the resonances of the protons of the heme are shifted well outside the 0 -10-ppm region of the protein NMR spectrum (38, 49 -51, 61, 62). The similarity of the relative intensities and chemical shifts of the heme resonances of recombinant house fly cyt b 5 to those of other cyt b 5 s (38,43,47,48), summarized in Table II, is striking. This finding indicates that the shape of the heme pocket of recombinant house fly cyt b 5 is similar to that of other cyt b 5 s. The small differences in chemical shifts represent only very minor changes in the orientation of the heme group with respect to the planes of the histidine ligands (62).…”

“…Synthetic cyt b 5 genes and natural cDNAs for cyt b 5 have been expressed previously in E. coli, either constitutively (46,53) or under the control of lacZ (54,55) or T7 promoter (40,43,56). We have expressed the cDNA of house fly cyt b 5 in the protease deficient E. coli strain BL21, under control of the double cassette of the strong synthetic isopropyl-␤-D-thiogalactopyranoside-inducible Tac promoter.…”