Dimethyl propionate ester heme-containing cytochrome b5: structure and stability.
A derivative of rat microsomal cytochrome b5, obtained by substitution of the native heme moiety with protoporphyrin IX dimethyl ester, has been characterized by 1H and 15N 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 methyls, as well as the directions of the rhombic axes of the magnetic susceptibility tensor, indicate a small difference in the orientation of the imidazole planes of the histidine axial ligands. The solution structure was determined by using 1,303 meaningful NOEs and 241 pseudocontact shifts, the latter being derived from the native reduced protein. A family of 40 energy-minimized conformers was obtained with average RMSD of 0.56+/-0.09 A and 1.04+/-0.12 A for backbone and heavy atoms, respectively, and distance and pseudocontact shift penalty functions of 0.50+/-0.07 A2 and 0.51+/-0.02 ppm2. The structure shows some changes around the cavity and in particular a movement of the 60-70 backbone segment owing to the absence of two hydrogen bonds between the Ser64 backbone NH and side-chain OH and the carboxylate oxygen of propionate-7, present in the native protein. The analysis of the NMR spectra in the presence of unfolding agents indicates 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 connecting propionate-7 to Ser64 in the native protein. The available data on the reduction potential and the electron transfer rates are discussed on the basis of the present structural data.
Banci
L
L
Magnetic Resonance Center, University of Florence, Italy.
Bertini
I
I
Branchini
B R
BR
Hajieva
P
P
Spyroulias
G A
GA
Turano
P
P
D000595:Amino Acid Sequence; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008280:Magnetics; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding
D008665:Metalloporphyrins; C445099:dimethyl propionate ester heme-containing cytochrome b5; D006418:Heme; D015786:Cytochromes b5; D019791:Guanidine