Characterization of Cytochrome b5 Reconstituted with a Ferric Chlorin and a Ferric Oxochlorin

Susan A. Martinis, Charlika Sotiriou, Chi K. Chang, Stephen G. Sligar

Research output: Contribution to journalArticle

Abstract

The role of the electronic properties of the heme group of rat cytochrome b5 in biological electron transfer was investigated by substituting chlorin analogues for the native protoporphyrin IX prosthetic group. The resultant purified proteins displayed physical and chemical properties distinct from those of the native enzyme. Optical spectroscopy of the ferric chlorin substituted cytochrome b5 revealed a blue-shifted Soret at 404 nm and a band at 586 nm characteristically red-shifted from the protohemin absorption band. The reduced, reconstituted protein displayed maxima at 406, 418, 455, 563, and 600 nm. The oxidized cytochrome b5 containing the oxochlorin analogue produced a red-shifted Soret with maxima at 338, 416, and 602 nm. The reduced species differed only in the visible region with absorption maxima at 508, 554, and 600 nm. Characterization by EPR spectroscopy of the oxochlorin-substituted cytochrome b5 yielded g values of 2.566, 2.375, and 1.756 and respective axial ∆/λ and rhombic V/λ components of 2.857 and 3.287, indicating significant electronic distortion in the chlorin ring and an increase in electron donation from the axial histidine ligands. A decrease in the reduction potential of 52 ± 5 mV (50 mM KPi, pH 7.0, 25 °C) for the chlorin-reconstituted cytochrome b5 was determined with respect to that of native cytochrome b5. The reduction potential for the oxochlorin-containing cytochrome b5 was unchanged from that of the native system. Both of the reconstituted proteins were found to be capable of transferring electrons to cytochrome c in a reconstituted system dependent on NADH and cytochrome b5 reductase, thus simulating the activity of native cytochrome b5.

Original languageEnglish (US)
Pages (from-to)879-884
Number of pages6
JournalBiochemistry
Volume28
Issue number2
DOIs
StatePublished - Jan 1 1989

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Cytochromes b5
Electrons
Spectrum Analysis
Cytochrome-B(5) Reductase
Hemin
Proteins
ferric oxochlorin
ferric chlorin
Prosthetics
Cytochromes c
Heme
Histidine
Electronic properties
Chemical properties
Paramagnetic resonance
Rats
Absorption spectra
Physical properties
Spectroscopy
Ligands

ASJC Scopus subject areas

  • Biochemistry

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Characterization of Cytochrome b5 Reconstituted with a Ferric Chlorin and a Ferric Oxochlorin. / Martinis, Susan A.; Sotiriou, Charlika; Chang, Chi K.; Sligar, Stephen G.

In: Biochemistry, Vol. 28, No. 2, 01.01.1989, p. 879-884.

Research output: Contribution to journalArticle

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