UNUSUAL BEHAVIOR OF A HEME IN A TETRAHEME PROTEIN, CYTOCHROME-C(3) FROM DESULFOVIBRIO-VULGARIS MIYAZAKI,F, IN THE REDUCTION PROCESS

A combination of two-dimensional (2D) total correlation spectroscopy ( TOCSY) and nuclear Overhauser enhancement and exchange spectroscopy (N OESY) has been used to assign the heme proton signals of cytochrome c( 3) from Desulfovibrio vulgaris Miyazaki F in the fully reduced state. The sequence-specific assignment of the four hemes was achieved by fol lowing chemical exchange connectivities of the heme methyl signals fro m the fully oxidized to fully reduced states in 2D exchange spectra. C ombining the results by one-dimensional saturation transfer and 2D exc hange experiments, the chemical shifts of the 14 heme methyl signals o ut of 16 were determined for the five macroscopic oxidation states. Th e result showed that two methyl signals of heme 3 behave unusually in terms of the intermediate chemical shifts. The assignment was also mad e for aromatic residues except for Phe20 and eight-coordinated histidi nes. On the basis of these assignments and nuclear Overhauser effects among these signals, the heme architectures in different states were d iscussed. (C) 1997 Elsevier Science S.A.