Dimethyl propionate ester heme-containing cytochrome b(5): structure and stability

A derivative of rat microsomal cytochrome b(5), obtained by substitution of the native heme moiety with protoporphyrin IX dimethyl ester, has been cha racterized by H-1 and N-15 NMR spectroscopy. Besides the two usual A and B forms, which depend on the orientation of the heme in the prostethic group cavity, two other minor forms have been detected which presumably indicate different conformations of the vinyl side chains. The shifts of the heme me thyls, as well as the directions of the rhombic axes of the magnetic suscep tibility tensor, indicate a small difference in the orientation of the imid azole planes of the histidine axial ligands. The solution structure was det ermined by using 1303 meaningful NOEs and 241 pseudocontact shifts, the lat ter being derived from the native reduced protein. A family of 40 energy-mi nimized conformers was obtained with average RMSD of 0.56 +/-0.09 Angstrom and 1.04 +/-0.12 Angstrom for backbone and heavy atoms, respectively, and d istance and pseudocontact shift penalty functions of 0.50 +/-0.07 Angstrom (2) and 0.51 +/-0.02 ppm(2). The structure shows some changes around the ca vity and in particular a movement of the 60-70 backbone segment owing to th e absence of two hydrogen bonds between the Ser64 backbone NH and side-chai n OH and the carboxylate oxygen of propionate-7, present in the native prot ein. The analysis of the NMR spectra in the presence of unfolding agents in dicates that this protein is less stable than the native form. The decrease in stability may be the result of the loss of the two hydrogen bonds conne cting propionate-7 to Ser64 in the native protein. The available data on th e reduction potential and the electron transfer rates are discussed on the basis of the present structural data.