The solution structure of the major form of the reduced soluble fragment of rat microsomal cytochrome b, has been solved through 'H-NMR spectroscopy. The protein contains 98 amino acids. Proton assignment was available for residues 1-94, except 90 [Guiles, R. D., Basus, V. J., Kuntz, I. D. & Waskell, L. (1992) Biochemistry 31, 11 365-11 3751 and has been confirmed. From 1722 NOES, of which 1203 were found to be meaningful, a family of 40 energy-minimized structures has been obtained with average backbone rmsd (for residues 5-89) of 0.078-tO.018 nm and average target function of 0.0045 nm', no distance violations being larger than 0.029 nm. The structure has been compared with the X-ray structure of the oxidized rat mitochondria1 isoenzyme and with that of the highly similar bovine microsomal isoenzyme in the oxidized form. The analysis of the elements of secondary structure is instructive in terms of their stability and of their occurrence in related structures, and of the capability of NMR and X-ray spectroscopy to observe them. Some detailed structural variations are noticed among the solved structures of the various isoenzymes and between solid and solution. The structural features in solution of the residues proposed to be involved in protein-protein recognition are found to be largely conserved with respect to the solid state.Keywords: cytochronie b,; protein recognition; solution structure; secondary structure.Cytochrome b, is a low-spin hemoprotein that acts as an electron-transfer mediator in numerous redox systems [I]. Different redox partners have been reported for cytochrome b, in vivo. In the process of fatty acid desaturation it interacts with NADH-cytochrome b, reductase [2, 31, and a role of cytochrome b, as electron donor to cytochrome P-450 has been proposed [4, 51. The protein is normally membrane bound, although a soluble form is found in erythrocytes, where its physiological role is that of reducing methemoglobin [6]. The membrane-bound form may be solubilized by incubation with proteolytic enzymes, such as trypsin. The soluble fragment of cytochrome b, still contains the heme and retains activity with respect to protein recognition and electron transfer [7]. It is well known that the soluble fragment of cytochrome b, interacts with cytochrome c, and that the reaction between the two is fast [7]. Such a complex has been an important model for the study of long-range biological electron transfer and, more generally, of the mechanisms of protein-protein recognition and interaction, mainly due because the crystallographic structures of the two individual components have been available since 1971 [8, 91. The interaction of cytochrome b, with cytochrome c has been studied extensively through a number of biochemical and biophysical methods [lo]. In particular, the small size of the two proteins allowed the investigation of the complex of the two through NMR spectroscopy as early as 1983 (111.The NMR investigation of cytochrome b, is significant in the frame of the current debate on the differences ...

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