Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c.

N Osheroff,B.A Feinberg,E Margoliash,M Morrison

doi:10.1016/s0021-9258(17)41031-3

Abstract

Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself. This conformationally intact derivative reacts with the succinate-cytochrome c reductase and the cytochrome c oxidase systems of beef mitochondrial particle preparations indistinguishably from the unmodified protein, showing that the region including tyrosine 74 is not involved in these enzymic electron transfer functions of the protein. The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74.

Highlights

Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself
The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74
Tyrosyl residue 74 is present in all eukaryotic cytochromes c so far examined except for the proteins from Crithidia oncopelti [71] and Humicola languinosa

Summary

74 Cytochrome c

67 and 74 [31] with horse cytochrome c, and similar mixtures of derivatized tyrosines. To investigate the role of surface tyrosyl residues, horse cytochrome c was subjected to iodination by lactoperoxidase [40]. Since this enzyme catalyzes the iodination within its active center, the exposed tyrosine residues are most readily attacked. 74 cytochrome c was found to be conformationally intact This made it possible for the first time to examine the functional role of a surface tyrosine and identify its contributions to the circular dichroic spectra of the protein. This residue was of interest because of its postulated role in a proposed pathway for the reduction of the protein [6, 41]. Preliminary accounts of some of this work have appeared [39, 42]

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DISCUSSION