Neil E. Mackenzie's research while affiliated with The University of Arizona and other places

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.

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).

It was shown by Koshy et al. [1990, Proc. Natl Acad. Sci USA, 87, 8697-8701; 1994, Biochem. J., 299, 347-350] that the substitution of proline 30 by alanine (P30A) of Drosophila melanogaster and rat cytochromes c exhibited decreased stabilities in both the heme iron-methionine sulfur (Fe-S) bond and overall protein conformation. Now we have found that the stability properties of the equivalent mutant of Rhodobacter capsulatus cytochrome c2 (P35A) are somewhat different. Based on optical and NMR spectroscopies, the Rb.capsulatus P35A alkaline transition (pKalk) was found to be unchanged with respect to the wild type, suggesting that the mutation in Rb.capsulatus cytochrome c2 has little effect on the stability of the Fe-S bond. However, Rb.capsulatus conformational stability was found to be decreased by 1.6 kcal/mol in the oxidized state. The difference in the stability properties of the equivalent proline to alanine substitutions in various species underscores the importance of studying mutations in more than one species before drawing generalizations about the role of conserved residues in protein structure and function.

In spite of marginal sequence homology, cytochrome c2 from photosynthetic bacteria and the mitochondrial cytochromes c exhibit some striking structural similarities, including the tertiary arrangement of the three main helices. To compare the folding mechanisms for these two distantly related groups of proteins, equilibrium and kinetic measurements of the folding/unfolding reaction of cytochrome c2 from Rhodobacter capsulatus were performed as a function of guanidine hydrochloride (GuHCl) concentration in the absence and presence of a stabilizing salt, sodium sulfate. Quenching of the fluorescence of Trp67 by the heme was used as a conformational probe. Kinetic complexities due to non-native histidine ligation are avoided, since cytochrome c2 contains only one histidine, His17, which forms the axial heme ligand under native and denaturing conditions. Quantitative kinetic modeling showed that both equilibrium and kinetic results are consistent with a minimal four-state mechanism with two sequential intermediates. The observation of a large decrease in fluorescence during the 2-ms dead-time of the stopped-flow measurement (burst phase) at low GuHCl concentration, followed by a sigmoidal recovery of the initial amplitude toward the unfolding transition region, is attributed to a well-populated compact folding intermediate in rapid exchange with unfolded molecules. A nearly denaturant-independent process at low GuHCl concentrations reflects the rate-limiting conversion of a compact intermediate to the native state. At high GuHCl concentrations, a process with little denaturant dependence is attributed to the rate-limiting Met96-iron deligation process during unfolding, which is supported by the kinetics of imidazole binding. The strong GuHCl-dependence of folding and unfolding rates near the midpoint of the equilibrium transition is attributed to destabilization of each intermediate and their transition states in folding and unfolding. Addition of sodium sulfate shifts the rate profile to higher denaturant concentration, which can be understood in terms of the relative stabilizing effect of the salt on partially and fully folded 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.

Comparative analysis of the 1H-NMR spectra of human insulin shows that in the presence of the allosteric ligand, phenol, the tertiary structure of the protein is altered as evidenced by the decreased rate of amide hydrogen-deuterium exchange. In particular, exchange of amide protons in residues of the B-chain helix (B9-B20) are significantly affected suggesting either a stabilization of this helix or a reduction in the solvent accessibility of the helix in the R-state. This paper exemplifies the exchange rates of two amides (ValB18 and TyrB16) from this helix which decrease by approximately 400-fold as a result of this ligand induced conformational transition.

The material balance for the biotransformation of grindelic acid by Aspergillus niger was determined along with the isolation of two novel dimers identified as methyl-8α-hydroxy-grindelate-7β-O-7′β-ether hydrate and methyl-3β-acetoxy-8α-hydroxy-grindelate-7β-O-7′β-ether hydrate. Also identified were the known 19-hydroxy-grindelic acid and the novel 6α,17-dihydroxy-grindelic acid. All compounds were isolated as their methyl ester acetate derivatives. The known 3β-hydroxy-7α,8α-epoxy grindelic acid was the sole product formed when 3β-hydroxy-grindelic acid was used as the substrate for biotransformation.

Acrylic acid (AA) and its esters are used extensively for the production of a variety of polymers. Despite their ubiquitous nature, little has been reported on the metabolism of the parent acid. The metabolites of AA may be volatile, unstable, polar, and thus difficult to isolate. Therefore, 13C NMR was used to help identify and compare directly the urinary metabolites of both 99% 13C-enriched AA and propionic acid (PA). Male Sprague-Dawley rats received [1,2,3-13C]AA (400 mg/kg in water p.o.) or an equimolar dose of [1,2,3-13C]propionate together with a radioactive tracer, [2,3-14C]AA, or [1-14C]propionate, respectively, and excreta were collected for 72 hr. For both acids, expiration of 14CO2 was the major route of elimination of radiolabel (approximately 80%). Approximately 6% of the dose was excreted in the urine. Urine was analyzed directly using proton-decoupled 13C and two-dimensional 13C homonuclear correlated NMR spectroscopy. The urine from AA-treated rats revealed major signals, the intensity of which was time-dependent, from at least five 13C-enriched metabolites of AA. Signals have been assigned to 3-hydroxypropionic acid, N-acetyl-S-(2-carboxyethyl)cysteine, and N-acetyl-S-(2-carboxyethyl)cysteine-S-oxide by comparison with spectra of authentic standards. No unchanged AA was detected. In contrast, the spectra of urine from a propionate-treated rat revealed only a few minor 13C-enriched signals that were assigned to methylmalonic acid. No unchanged PA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of tryptophan by promastigotes of Leishmania donovani donovani was investigated in cells suspended in a simple buffer solution supplemented with glucose. Metabolites from supernatant and lysed cell pellets were analyzed by capillary gas liquid chromatography and 13C nuclear magnetic resonance spectroscopy, with structural confirmation by gas liquid chromatography-mass spectrometry. Tryptophan does not appear to serve as a carbon energy source for L. d. donovani promastigotes since parasites could survive for only short periods in buffer containing tryptophan without glucose, levels of tricarboxylic acid cycle intermediates remained unchanged in the presence of added tryptophan and label from [13C]tryptophan was not detected in any of the intermediates. Leishmania d. donovani catabolized L-tryptophan via aminotransferase and aromatic lactate dehydrogenase reactions to form one major end product, indole-3-lactic acid. The activity of aromatic lactate dehydrogenase required manganese and was NADH-dependent in these organisms that lack lactate dehydrogenase. Promastigotes taken from the mid-log stage of growth produced higher concentrations of indole-3-lactic acid than those from the stationary stage. Conservation of a similar tryptophan catabolic pathway among four Leishmania species suggests the pathway is physiologically important to the parasites themselves.