Functional role of heme ligation in cytochrome c. Effects of replacement of methionine 80 with natural and non-natural residues by semisynthesis.
The nature of the axial ligation to heme iron has been suggested to be the major determinant of the oxidation-reduction potential of a particular cytochrome, but natural cytochromes that vary significantly in E'm invariably differ from one another in many ways. We proposed to clarify this issue by engineering many different ligation patterns within the same basic molecule, mitochondrial cytochrome c. Since many of the potentially informative substitutions require non-coded amino acids, semisynthesis was the approach we chose, and solid-phase peptide synthesis was used to make a set of nin 39-residue peptides that have been incorporated by autocatalytic fragment religation into the structure of horse cytochrome c. An additional two analogues modified at this position were made by chemical modification of the whole protein. As well as looking at the effect on reduction potential, we examined the effect of varying the ligand sphere on the efficiency of the autocatalytic fragment religation reaction, on the conformation of cytochrome c, on its spectroscopic properties, and in promoting electron transfer between heme c and other redox centers. Substitute residues were chosen to put sulfur, selenium, oxygen, and nitrogen, or even no ligating atom at all in the place of methionine sulfur. We found both subtle and dramatic alterations in spectral properties, which were informative about changes in internal structure and stability brought about by the modifications and which may be useful in identifying novel natural ligation patterns. An unexpected finding was that alanine 80 cytochrome c acquires a hemoglobin-like spectrum, and binds O2 most effectively. Reduction potential changes of greater than 300 mV with nitrogen, greater than 400 mV with oxygen, and greater than 300 mV with thiol sulfur ligation were observed, confirming that variation of the ligand sphere is indeed the most effective way in which the protein coat may modulate the potential of the redox center it encloses. Finally, we obtained more evidence that this axial ligand plays an active role in electron transfer and discovered that histidine could be even more effective in this role.
Wallace
C J
CJ
Department of Biochemistry, Dalhousie University Halifax, Nova Scotia, Canada.
Clark-Lewis
I
I
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D006736:Horses; D007501:Iron / Q000378:metabolism; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002412:Cations; D003574:Cytochrome c Group; D006418:Heme; D008715:Methionine; D007501:Iron; D010100:Oxygen