pH-induced changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: an 15N NMR study.

Abstract

The 15N-enriched ferrocytochrome c2 from Rhodospirillum rubrum was studied by '5N NMR at different solvent pH values. The m bility and chemical shift of the N-terminal glutamic acid (35.i.4 ppm at pH 5.1) were found to depend on pH. It was least mobile between pH 8 and 9.0, which is explained in terms of pH-dependent conformational changes and formation of salt linkages and/or hydrogen bonds. The resonances of the lysine side chains are centered around 341.7 ppm at low pH and move upfield with pH by about 8.4 ppm with pK. values of 10.8. The exchange rates of the eNH protons are lowest near their pK. values. The protein is very stable in the pH range between 4.9 and 10.0 but unfolds abruptly at pH 10.5-11. Denaturation was verified by the measurement of several parameters by NMR. The renaturation of the protein demonstrates that the folding begins with reformation of heme coordination and establishment of a hydrophobic core, followed by positioing of side chains and peptide backbones linking the nucleation centers. The repositioning processes had time scales of minutes to hours in contrast to the reported values of seconds in some studies.The cytochromes c2 are a class of electron-transfer proteins found in the non-sulfur purple photosynthetic bacteria. One characteristic of the cytochromes is their redox potential, which, for cytochrome c2, has been found to be very pH dependent (1,2). Ionizable groups in the protein include the ligand histidine and a non-ligand histidine, a single invariant tryptophan, various acidic residues, the N and C termini, and the two propionic acid substituents of the heme. Among the charged groups of the protein, the lysine side-chain groups on the surface of the protein which appear to be very mobile (3) are proposed to play an essential role in electron transfer (4). Local motion of other groups and segmental motion of the N-terminal a-helix are thought to be important characteristics of the protein's structure (5).Recent studies on cytochrome structure include one-and two-dimensional proton NMR studies of the mitochondrial cytochrome (6-9). These studies, which are approaching complete assignment of the 1H NMR spectrum of the protein, follow an abundant literature.Although the structures of cytochromes are well studied by x-ray crystallography (10-12) and are known to be conserved (13,14), their dynamic structures in solution and pH dependence are not well understood. In previous work (15), we have assigned the important 15N resonances and observed conformational changes of the reduced state of cytochrome c2 induced by changes in pH using 15N NMR. In the present paper, we report the observation of pH-induced denaturation and refolding of cytochrome c2. MATERIALS AND METHODSGrowth of Rhodospirlum rubrum. '5NH4Cl was obtained from MSD isotopes. The G-9 (blue-green) mutant of R. rubrum was kindly provided by P. F. Weaver (SERI). Growth media and conditions were as described (15). The 15N-enriched cytochrome c2 was purified from 15NH4CI-grown R. rubrum by standard methods (16,...