Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2.

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.

Structure and stability effects of the mutation of glycine 34 to serine in Rhodobacter capsulatus cytochrome c(2).

Gly 34 and the adjacent Pro 35 of Rhodobacter capsulatus cytochrome c(2) (or Gly 29 and Pro 30 in vertebrate cytochrome c) are highly conserved side chains among the class I c-type cytochromes. The mutation of Gly 34 to Ser in Rb. capsulatus cytochrome c(2) has been characterized in terms of physicochemical properties and NMR in both redox states. A comparison of the wild-type cytochrome c(2), the G34S mutation, and the P35A mutation is presented in the context of differences in chemical shifts, the differences in NOE patterns, and structural changes resulting from oxidation of the reduced cytochrome. G34S is substantially destabilized relative to wild-type (2.2 kcal/mol in the oxidized state) but similarly destabilized relative to P35A. Nevertheless, differences in terms of the impact of the mutations on specific structural regions are found when comparing G34S and P35A. Although available data indicates that the overall secondary structure of G34S and wild-type cytochrome c(2) are similar, a number of both perturbations of hydrogen bond networks and interactions with internal waters are found. Thus, the impact of the mutation at position 35 is propagated throughout the cytochrome but with alterations at defined sites within the molecule. Interestingly, we find that the substitution of serine at position 34 results in a perturbation of the heme beta meso and the methyl-5 protons. This suggests that the hydroxyl and beta carbon are positioned away from the solvent and toward the heme. This has the consequence of preferentially stabilizing the oxidized state in G34S, thus, altering hydrogen bond networks which involve the heme propionate, internal waters, and key amino acid side chains. The results presented provide important new insights into the stability and solution structure of the cytochrome c(2).

An optimized g-tensor for Rhodobacter capsulatus cytochrome c2 in solution: a structural comparison of the reduced and oxidized states.

The optimized g-tensor parameters for the oxidized form of Rhodobacter capsulatus cytochrome c2 in solution were obtained using a set (50) of backbone amide protons. Dipolar shifts for more than 500 individual protons of R. capsulatus cytochrome c2 have been calculated by using the optimized g-tensor and the X-ray crystallographic coordinates of the reduced form of R. capsulatus cytochrome c2. The calculated results for dipolar shifts are compared with the observed paramagnetic shifts. The calculated and the observed data are in good agreement throughout the entire protein, but there are significant differences between calculated and experimental results localized to the regions in the immediate vicinity of the heme ligand and the region of the front crevice of the protein (residues 44-50, 53-57, and 61-68). The results not only indicate that the overall solution structures are very similar in both the reduced and oxidized states, but that these structures in solution are similar to the crystal structure. However, there are small structural changes near the heme and the rearrangement of certain residues that result in changes in their hydrogen bonding concomitant with the change in the oxidation states; this was also evident in the data for the NH exchange rate measurements for R. capsulatus cytochrome c2.

Hymenolepis diminuta: nuclear magnetic resonance analysis of the excretory products resulting from the metabolism of D-[13C6]glucose.

Excretory products of the tapeworm Hymenolepis diminuta, fed D-[13C6]glucose in vitro for 90 min, were studied using 1H and 13C nuclear magnetic resonance spectroscopy. Signals due to lactate, succinate, acetate, and alanine were identified in the spectra. Several differently labeled species were present for these metabolites; the variations of higher concentration were a consequence of metabolic factors while those of lower concentration could be accounted for by residual 12C in the glucose. The two major labeled lactates, U-13C and 2,3-13C2, were in the ratio 2:1, respectively, and the three major labeled succinates, 1,2,2'-13C3,2,2'-13C2, and U-13C, were present in the ratio 20:10:3, respectively. The different species of labeled end products are related to the overall glucose metabolism of H. diminuta.