Structural changes in cytochrome c upon hydrogen-deuterium exchange

Abstract

The resonance Raman spectra of yeast ferri- and ferro-iso-1-cytochrome c dissolved in H2O and D2O are reported. Hydrogen exchange in the protein leads to distinct spectral changes of heme vibrational bands, particularly in the region between 670 and 710 cm-1 and at approximately 443 and approximately 450 cm-1. The latter two bands, which have previously been assigned to porphyrin modes including bending vibrations of the propionate side chains [Hildebrandt, P. (1991) J. Mol. Struct. 242, 379-395], reveal frequency shifts by up to 4 cm-1. These shifts are attributed to structural changes of the propionate groups caused by the energetic differences of the hydrogen and deuterium bonds between these substituents and the adjacent amino acid residues. The frequency shifts of the bands between 670 and 710 cm-1 most likely reflect structural differences of the tetrapyrrole macrocycle itself. Time-dependent experiments revealed that the hydrogen exchange processes associated with the changes in the resonance Raman spectra are complete in less than 15 min. The protons which are involved are those in the interior of the heme pocket as concluded by comparison with the exchange rate constants previously determined by NMR spectroscopy [Mayne, L., Paterson, Y., Cerasoli, D., & Englander, S. W. (1992) Biochemistry 31, 10678-10685]. These protons are part of a hydrogen bonding network including the amide protons of Asn-52, Met-80, and Lys-79, the side chain protons of Asn-52, Tyr-67, Thr-78, Trp-59, and Thr-49, and the water molecules 121 and 166.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Deuterium Substitution on Electron Transfer at Cytochrome c/SAM Interfaces

“…4 From our experiments, however, we can assign a lower time limit of about 2 h to any H/D exchange process that affects the rate in covalent assemblies. Considering the close similarity in the composition, 24,39 electrochemical properties, and electron transfer rate constants 17,21,22 of the two assemblies, this time limit seems probable for electrostatic assemblies as well.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] For example, adsorption to a pure -S(CH 2 ) m COOH or mixed -S(CH 2 ) m COOH/ -S(CH 2 ) n OH SAM via electrostatic interactions between cationic lysine residues on the protein and anionic carboxylate residues on the SAM ( Figure 1A) results in a situation that is similar to the interaction of cytochrome c with cytochrome c oxidase, 18 its biological electron transfer partner. Electrostatic interactions can be converted to amide bonds using a covalent cross-linking reagent such as EDC 19,20 or CMC, 17,21,22 as illustrated by Figure 1B, leading to an electron transfer complex in which large-scale rotational motion of the protein is restricted.…”

Enhanced Prediction Accuracy of Protein Secondary Structure Using Hydrogen Exchange Fourier Transform Infrared Spectroscopy

“…It has been suspected that structural complications might arise due to D 2 O, but, in fact, such isotopically induced structural changes in proteins have not been determined. Goto et al (77) did not find any noticeable deuteration effects on the protein folding of apomyoglobin, while Hildebrandt et al (52) did find an association between the spectral changes with structural modifications in the heme pocket of cytochrome c. However, these latter variations are unlikely to be equilibrium secondary structure changes. Effects due to pH have been noted before, especially on exchange rates (36,78,79); but as mentioned above, in this study we have maintained a consistency with previously published work for the sake of comparative analysis, so that pH effects were not considered.…”

“…Its chemical and physical mechanisms have been studied extensively (33)(34)(35)(36)(37)(38)(39)(40), and their impact has proven to be most valuable with deuterationsensitive spectroscopic methods such as IR (41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51), Raman (52), NMR (37,38,(53)(54)(55)(56)(57)(58)(59)(60)(61)(62), mass spectrometry (63)(64)(65)(66)(67)(68), and neutron diffraction (69).…”

Mechanisms and uses of hydrogen exchange

“…Hydrogen exchange can be measured at an unresolved level by tritium counting and by various spectroscopic methods (infrared [39,40], Raman [41], UV absorbance [42], NMR), at intermediate resolution by tritium exchange labeling together with fragment-separation methods [14,43,44], and at the ultimate resolution of individual protons by NMR methods and neutron crystallography [21]. Recent applications of mass spectrometry also hold promise [45-50,51 • ].…”