Abstract
The significance of the exposed haem edge in cytochrome c was directly probed by chemically modifying the partially exposed haem propionate in the crevice region around residues threonine-78 and threonine-49. Reaction of tuna heart cytochrome c with a water-soluble carbodi-imide at pH 3.7 in the absence of any added nucleophilic base leads to the covalent addition of substituted N-acylureas to the protein at two sites. One site has been shown to be a haem propionate by isotope-tracer and i.r.-spectral analysis of haem purified from the apoprotein. The other site is aspartial acid-62 on the back of the molecule. The modified cytochrome c demonstrates abnormal properties, including auto-oxidizability, a reduction potential of + 105mV, a reversible transition to a high-spin species below pH 5.3, no 695 nm charge-transfer band in the ferric state and abnormal binding to mitochondrial membranes. The derivative does react with cytochrome oxidase in deoxycholate-treated submitochondrial particles or in purified preparations with a specific activity of 43-65% compared with that obtained with native cytochrome c. The results are consistent with the view that an intact haem crevice is essential for normal values for physiochemical characteristics, but the significant residual enzymic activity suggests that the electron-transfer interface and/or the cytochrome oxidase-binding site cannot be localized solely in the region of the exposed haem propionate.