Nuclear magnetic resonance studies of slowly exchanging peptide protons in cytochrome c in aqueous solution.

Abstract

The slowly exchanging protons in oxidized and reduced horse heart cytochrome c (D20, uncorrected pH meter reading 6.5, room temperature) have been monitored by recording the 270 and 360 MHz proton nuclear magnetic resonance spectra of the reduced protein between 5 and 11 parts per million downfield from 2,Aimethyl-2-silapentane-5-sulfonate.There are about 12 well-resolved exchangeable resonances between 7.9 and 10.1 parts per million and the experimental chemical shift data suggest that they originate from the same residues in both oxidation states. These resonances correspond to 19-22 protons in the reduced protein and a lesser number due to partial exchange in the oxidized protein. The base catalysis of the exchange in both oxidation states was used to demonstrate the sequential exchange of these well-resolved resonances in D20. Alternatively, the temperature dependence of the exchange at a fixed pH was used to differentiate and monitor separately the slower and the slowest exchanging resonances as a function of time. The different rates of exchange among the slowly exchanging peptide protons provide evidence against a resistant cluster of protons that exchange together. Cytochrome c has been studied extensively by chemical and physical techniques with the eventual goal of understanding its role in coupled electron transport (1, 2). X-ray crystallographic studies of the oxidized and reduced cytochrome c have added considerably to our knowledge of the polypeptide conformation and heme group contacts in the crystal (3, 4). Physiological mechanisms have been proposed for the oxidoreduction of cytochrome c based on the available x-ray data on the cytochrome class of proteins (5, 6).Nuclear magnetic resonance (NMR) spectroscopic studies have elucidated many aspects of the cytochrome c -structure in solution (7-12, and Exchange Studies on the Oxidized Protein. Oxidized cytochrome c was dissolved in H20, the pH was adjusted to 6.5, and the solution was lyophilized. The lyophilized material was dissolved in D20 to an 8 mM protein concentration and the pH (uncorrected pH meter reading) was raised to the desired value with NaOD. The samples were heated in a water bath at a given temperature and the aliquots were removed as a function of time. The oxidized samples were reduced with dithionite and the NMR spectra were recorded on the reduced cytochrome c in D20 solution.Exchange Studies on the Reduced Protein. Oxidized cytochrome c was dissolved in H20 solution, the pH was adjusted to 6.5, and the protein was reduced with dithionite. The reduced protein in H20 at pH 6.5 was lyophilized, then dissolved in D20 solution to an 8 mM protein concentration, and the uncorrected pH meter reading was changed to the desired value with NaOD. The samples were heated in the water bath at the desired temperature and aliquots of the reduced protein were removed as a function of time to record the NMR spectra. Proton NMR spectra

Transparent Window Vibrational Probes for the Characterization of Proteins With High Structural and Temporal Resolution

“…However, both X-ray and NMR solution structures clearly indicated that the two states are virtually identical . Thus, the observed differences in rates of proteolysis, − radius of gyration, ligand binding, − and H/D isotope exchange, − along with the negative entropy of reduction, , led to the conclusion that the oxidized state was more flexible and thus sampled a wider range of conformational substates than the reduced protein . The increased rigidity of the reduced protein was also soon linked to an increase in the strength of the Met80 S–Fe bond relative to the oxidized state .…”

Protein Compressibility, Dynamics, and Pressure

“…¶ Kharakoz and Mkhitaryan (1986). Ulmer and Kägi (1968); Patel and Canuel (1976). **Determined by comparing the separately prepared samples of the two protein forms (a large systematic error is possible).…”

Amide proton exchange rates of oxidized and reduced saccharomyces cerevisiae iso‐1‐cytochrome c

“…The term k3 is the rate constant for exchange once the labile site contacts the catalyst. This second step is primarily base catalyzed above pH 4 (McDonald & Phillips, 1973;Patel & Canuel, 1976;Englander & Kallenbach, 1984), but water itself can also react directly (Gregory et al, 1983). Acid catalysis occurs at lower pH (Perrin, 1989).…”