Characterization of cytochrome b5 reconstituted with a ferric chlorin and a ferric oxochlorin.
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, 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 delta/lambda and rhombic V/lambda 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 degrees 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 stimulating the activity of native cytochrome b5.
Martinis
S A
SA
Department of Biochemistry, University of Illinois, Urbana-Champaign 61801.
Sotiriou
C
C
Chang
C K
CK
Sligar
S G
SG
D000818:Animals; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000494:pharmacology*; D008665:Metalloporphyrins / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011524:Protoporphyrins / Q000378:metabolism; D051381:Rats; D013053:Spectrophotometry
D003573:Cytochrome b Group; D005290:Ferric Compounds; D008665:Metalloporphyrins; D011524:Protoporphyrins; C059625:ferric oxochlorin; C059624:ferric chlorin; D015786:Cytochromes b5; C028025:protoporphyrin IX