MUTAGENESIS OF HISTIDINE-26 DEMONSTRATES THE IMPORTANCE OF LOOP-LOOP AND LOOP-PROTEIN INTERACTIONS FOR THE FUNCTION OF ISO-1-CYTOCHROME-C

In yeast iso-l-cytochrome c, the side chain of histidine 26 (His26) at taches omega loop A to the main body of the protein by forming a hydro gen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra-loop interaction through a h ydrogen bond to the backbone amide of asparagine 31. To investigate th e importance of loop-protein attachment and intra-loop interactions to the structure and function of this protein, a series of site-directed and random-directed mutations were produced at His26. Yeast strains e xpressing these variant proteins were analyzed for their ability to gr ow on non-fermentable carbon sources and for their intracellular produ ction of cytochrome c. While the data show that mutations at His26 lea d to slightly decreased intracellular amounts of cytochrome c, the lev el of cytochrome c function is decreased more. The data suggest that c ytochrome c reductase binding is affected more than cytochrome c oxida se or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein 's ability to bind redox partners.