Noncovalent binding of heme induces a compact apocytochrome c structure.
Mitochondrial holocytochrome c contains heme that is covalently attached to the protein in a reaction catalyzed by the enzyme cytochrome c heme lyase. In the absence of heme, apocytochrome c, the precursor to holocytochrome c, is unfolded. We find that purified apocytochrome c binds noncovalently to heme. Binding is accompanied by changes in the optical absorption spectrum of heme and by quenching of the tryptophan fluorescence of the protein. The affinity of apocytochrome c for heme, as well as the stoichiometry of binding, appears to depend on whether or not cyanide is present and on the oxidation state of heme. Under reducing conditions, in the presence of cyanide, the association appears to be 1:1, with a binding constant of about 10(7) M-1. Under oxidized conditions, there may be multiple hemes bound per molecule of apocytochrome c. Upon binding to heme, apocytochrome c exhibits a mobility similar to that of holocytochrome c in gel filtration chromatography and velocity gradient ultracentrifugation, indicating that the heme-protein complex adopts a structure that is almost as compact as that of holocytochrome c. Changes in the circular dichroism spectrum of apocytochrome c are consistent with an increase in the alpha-helical content of the protein on binding heme. The compact structure of the noncovalent heme-apocytochrome c complex may represent an intermediate in the de novo folding of cytochrome c.
Dumont
M E
ME
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Corin
A F
AF
Campbell
G A
GA
D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045304:Cytochromes c; D006418:Heme / Q000378:metabolism*; D006736:Horses; D007501:Iron / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012260:Ribonucleases / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D003574:Cytochrome c Group; D006418:Heme; D045304:Cytochromes c; D007501:Iron; D012260:Ribonucleases