Axial ligands of chloroplast cytochrome b-559: Identification and requirement for a heme-crosslinked polypeptide structure

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Abstract
Optical, resonance Raman, and EPR spectroscopies were used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low-potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its 5th and 6th coordination positions by histidine N. EPR data for the purified spinach cytochrome are in good agreement with those determined by Bergstrom and Vanngard for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift interpreted as arsing from a deviation of the planes of the 2 axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low- and high-potential forms. Recent biochemical and molecular biological data show that 2 polypeptides, 1 with 83 residues and a 2nd with 39 residues, most likely constitute the protein portion of the cytochrome. Each of the polypeptides contains only a single histidine. Thus, to provide the bis(histidine) axial ligation required by the physical data, the heme may act as a cross-linker by coordinating histidines from 2 different polypeptide chains.