Nitrogen isotopic discrimination in dietary amino acids: The threonine anomaly.

RATIONALE: The "Threonine Anomaly" relates to an observation made 25 years ago on the change in Thr nitrogen isotopic ratio in mammalian metabolism. Unlike all other amino acids, Thr in body protein is found to be depleted (rather than enriched) in 15 N relative to dietary Thr. Interpreting isotopic discrimination has become a useful source of ecological and palaeodietary information and it is desirable that the underlying processes are understood. METHODS: The principal enzyme of threonine catabolism, suggested to be responsible for the anomaly, threonine dehydratase, was prepared from rat liver. A time course of incubation of the enzyme with pure threonine was followed, and samples of residual threonine prepared for isotopic analysis by combustion in an automated carbon and nitrogen analyser coupled to a continuous flow isotope ratio mass spectrometer. RESULTS: We show experimentally, in vitro, that the enzymic reaction catabolising Thr cannot be responsible for its 15 N depletion. Plots of delta 15 N against both reaction time course and percentage completion show in fact an accelerating enrichment. CONCLUSIONS: A previously advanced suggestion that the unique catabolic mechanism for threonine was responsible for the anomalous depletion in 15 N is clearly not the case. We therefore offer alternative explanations, based on threonine's role at an organismal rather than cellular level. Copyright © 2016 John Wiley & Sons, Ltd.

Cytochrome c impaled: investigation of the extended lipid anchorage of a soluble protein to mitochondrial membrane models.

Cyt c (cytochrome c) has been traditionally envisioned as rapidly diffusing in two dimensions at the surface of the mitochondrial inner membrane when not engaged in redox reactions with physiological partners. However, the discovery of the extended lipid anchorage (insertion of an acyl chain of a bilayer phospholipid into the protein interior) suggests that this may not be exclusively the case. The physical and structural factors underlying the conformational changes that occur upon interaction of ferrous cyt c with phospholipid membrane models have been investigated by monitoring the extent of the spin state change that result from this interaction. Once transiently linked by electrostatic forces between basic side chains and phosphate groups, the acyl chain entry may occur between two parallel hydrophobic polypeptide stretches that are surrounded by positively charged residues. Alteration of these charges, as in the case of non-trimethylated (TML72K) yeast cyt c and Arg91Nle horse cyt c (where Nle is norleucine), led to a decline in the binding affinity for the phospholipid liposomes. The electrostatic association was sensitive to ionic strength, polyanions and pH, whereas the hydrophobic interactions were enhanced by conformational changes that contributed to the loosening of the tertiary structure of cyt c. In addition to proposing a mechanistic model for the extended lipid anchorage of cyt c, we consider what, if any, might be the physiological relevance of the phenomenon.

Probing the role of the conserved beta-II turn Pro-76/Gly-77 of mitochondrial cytochrome c.

The loop segment comprising residues 70-84 in mitochondrial cytochrome c serves to direct the polypeptide backbone to permit the functionally required heme Fe - S (Met-80) co-ordination. The primary sequence here is highly conserved, which is something rarely observed in surface loop segments and suggests that its purpose is more complex than its obvious structural role. The beta-II turn formed by Pro-76 and Gly-77 is postulated to be key to the redirection of the peptide backbone required to execute the loop. We assessed the importance of Pro-76 and Gly-77 by mutating 1 or both of these residues to alanine such that the range of allowable dihedral angles was altered, and this resulted in significant changes in physicochemical properties and biological activities. We observed structural perturbations using circular dichroism spectroscopy and thermal denaturation studies. Based on these changes, we propose that the Pro-76/Gly-77 beta-II turn precisely orients the 70s loop, not only to maintain the backbone orientation required for the formation of the axial heme ligand, but also to provide a complementary surface to physiological partners.

Static normal coordinate deformations of the heme group in mutants of ferrocytochrome c from Saccharomyces cerevisiae probed by resonance Raman spectroscopy.

The function of heme proteins is, to a significant extent, influenced by the ligand field probed by the heme iron, which itself can be affected by deformations of the heme macrocycle. The exploration of this field is difficult because the heme structure obtained from X-ray crystallography is not resolved enough to unambiguously identify structural changes on the scale of 10(-2) A. However, asymmetric deformations in this order of magnitude affect the depolarization ratio of the resonance Raman lines assignable to normal vibrations of the heme group. We have measured the dispersion of the depolarization ratios of four structure sensitive Raman bands (i.e., nu4, nu11, nu21, and nu28) in yeast iso-1-ferrocytochrome c and its mutants N52V, Y67F, and N52VY67F with B- and Q-band excitation. The DPR dispersion of all bands indicates the presence of asymmetric in-plane and out-of-plane deformations. The replacement of the polar tyrosine residue at position 67 by phenylalanine significantly increases the triclinic B2g deformation, which involves a distortion of the pyrrole symmetry. We relate this deformation to changes of the electronic structure of pyrrole A, which modulates the interaction between its propionate substituents and the protein environment. This specific heme deformation is eliminated in the double mutant N52VY67F. The additional substitution of N52 by valine induces a tetragonal B1g deformation which involves asymmetric changes of the Fe-N distances and increases the rhombicity of the ligand field probed by the heme iron. This heme deformation might be caused by the elimination of the water-protein hydrogen-bonding network in the heme cavity. The single mutation N52V does not significantly perturb the heme symmetry, but a small B1g deformation is consistent with our data and the heme structure obtained from a 1 ns molecular dynamics simulation of the protein.

Optical band splitting and electronic perturbations of the heme chromophore in cytochrome C at room temperature probed by visible electronic circular dichroism spectroscopy.

We have measured the electronic circular dichroism (ECD) of the ferri- and ferro-states of several natural cytochrome c derivatives (horse heart, chicken, bovine, and yeast) and the Y67F mutant of yeast in the region between 300 and 750 nm. Thus, we recorded the ECD of the B- and Q-band region as well as the charge-transfer band at approximately 695 nm. The B-band region of the ferri-state displays a nearly symmetric couplet at the B0-position that overlaps with a couplet 790 cm-1 higher in energy, which we assigned to a vibronic side-band transition. For the ferro-state, the couplet is greatly reduced, but still detectable. The B-band region is dominated by a positive Cotton effect at energies lower than B0 that is attributed to a magnetically allowed iron-->heme charge-transfer transition as earlier observed for nitrosyl myoglobin and hemoglobin. The Q-band region of the ferri-state is poorly resolved, but displays a pronounced positive signal at higher wavenumbers. This must result from a magnetically allowed transition, possibly from the methionine ligand to the dxy-hole of Fe3+. For the ferro-state, the spectra resolve the vibronic structure of the Qv-band. A more detailed spectral analysis reveals that the positively biased spectrum can be understood as a superposition of asymmetric couplets of split Q0 and Qv-states. Substantial qualitative and quantitative differences between the respective B-state and Q-state ECD spectra of yeast and horse heart cytochrome c can clearly be attributed to the reduced band splitting in the former, which results from a less heterogeneous internal electric field. Finally, we investigated the charge-transfer band at 695 nm in the ferri-state spectrum and found that it is composed of at least three bands, which are assignable to different taxonomic substates. The respective subbands differ somewhat with respect to their Kuhn anisotropy ratio and their intensity ratios are different for horse and yeast cytochrome c. Our data therefore suggests different substate populations for these proteins, which is most likely assignable to a structural heterogeneity of the distal Fe-M80 coordination of the heme chromophore.

Functionally relevant electric-field induced perturbations of the prosthetic group of yeast ferrocytochrome c mutants obtained from a vibronic analysis of low-temperature absorption spectra.

We have measured the low temperature (T = 20 K) absorption spectra of the N52A, N52V, N52I, Y67F, and N52AY67F mutants of ferrous Saccharomyces cerevisiae (baker's yeast) cytochrome c. All the bands in the Q0- and Q(v)-band region are split, and the intensity distributions among the split bands are highly asymmetric. The spectra were analyzed by a decomposition into Voigtian profiles. The spectral parameters thus obtained were further analyzed in terms of the vibronic coupling model of Schweitzer-Stenner and Bigman (Schweitzer-Stenner, R.; Bigman, D. J. Phys. Chem. B 2001, 7064-7073) to identify parameters related to electronic and vibronic perturbations of the heme macrocycle. We report that the electronic perturbation is of B(1g) symmetry and reflects the heterogeneity of the electric field at the heme, that is, the difference between the gradients along the perpendicular N-Fe-N axis of the heme core. We found that all the investigated mutations substantially increase this electronic perturbation, so that the spectral properties become similar to those of horse heart cytochrome c. Moreover, the electronic perturbation was found to correlate nonlinearly with the enthalpy changes associated with the reduction of the heme iron. Group theoretical arguments are invoked to propose a simple model which explains how a perturbation of the obtained symmetry can stabilize the reduced state of the heme iron. Finally, vibronic coupling parameters obtained from the analysis of the Q(v)-band region suggest that the investigated mutations decrease the nonplanar deformations of the heme group. This finding was reproduced by a normal mode structural decomposition (NSD) analysis of the N52V and N52VY67F heme conformations obtained from a 1 ns molecular dynamics simulation. We argue that the reduced nonplanarity contributes to the stabilization of the reduced state.

Probing electrostatic interactions in cytochrome c using site-directed chemical modification.

This communication reports the generation of an electrostatic probe using chemical modification of methionine side chains. The alkylation of methionine by iodoacetamide was achieved in a set of Saccharomyces cerevisiae iso-1-cytochrome c mutants, introducing the nontitratable, nondelocalized positive charge of a carboxyamidomethylmethionine sulfonium (CAMMS) ion at five surface and one buried site in the protein. Changes in redox potential and its variation with temperature were used to calculate microscopic effective dielectric constants operating between the probe and the heme iron. Dielectric constants (epsilon) derived from deltadeltaG values were not useful due to entropic effects, but epsilon(deltadeltaH) gave results that supported the theory. The effect on biological activity of surface derivatization was interpreted in terms of protein-protein interactions. The introduction of an electrostatic probe in cytochrome c often resulted in marked effects on activity with one of two physiological partners: cytochrome c reductase, especially if introduced at position 65, and cytochrome c oxidase, if at position 28.

Phospholipid-cytochrome c interaction: evidence for the extended lipid anchorage.

Binding of cytochrome c (cyt c) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt c, revealing conformational changes in the protein. The exact mechanism of the interaction of fatty acids and acidic phospholipids with cyt c is unknown. Binding of cyt c to liposomes with high contents (mole fraction X > 0.7) of acidic phospholipids caused spectral changes identical to those due to binding of oleic acid. Fluorescence spectroscopy of a cyt c analog containing a Zn(2+) substituted heme moiety and brominated lipid derivatives (9,10)-dibromostearate and 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol demonstrated a direct contact between the fluorescent [Zn(2+)-heme] group and the brominated acyl chain. These data constitute direct evidence for interaction between an acyl chain of a membrane phospholipid and the inside of the protein containing the heme moiety and provide direct evidence for the so-called extended-lipid anchorage of cyt c to phospholipid membranes. In this mechanism, one of the phospholipid acyl chains protrudes out of the membrane and intercalates into a hydrophobic channel in cyt c while the other chain remains in the bilayer.