The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles.
Calculations of the redox potentials of the 2-/3-couples of [Fe4S*4Cys4] clusters in the iron-sulfur proteins Peptococcus aerogenes ferredoxin (PaFd), Azotobacter vinelandii ferredoxin I (AvFdI) and Chromatium vinosum high potential iron protein (CvHiPIP) based on the Protein Dipoles Langevin Dipoles (PDLD) method are reported. The structures of these proteins have been determined by X-ray crystallography; in the case of PaFd the structure has recently been revised due to a change in the sequence close to Cluster II. The large differences between the potentials of the [Fe4S*4Cys4] clusters of PaFd and AvFdI and the potential of the [Fe4S*4Cys4] cluster of CvHiPIP are successfully modeled and originate principally in differences in the configuration of main-chain amide groups near the clusters. The small difference between the potentials of PaFd and AvFdI is also satisfactorily modeled in the case of Cluster I of PaFd. Solvent dipoles close to the cluster in PaFd are an important contributor to its higher potential. The two X-ray structures of PaFd yield similar results for Cluster I of PaFd. In contrast, the results for Cluster II differ substantially; for reasons not yet clear, the recently revised structure leads to results in worse agreement with experiment.
The crystal structure of the His-175 -) Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (""5%), which, in view ofthe very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.The parameters required of the axial ligands for defining the diverse chemistry of heme enzymes are elusive. Peroxidases often contain a histidine ligand to the iron, whereas cysteine is found in monooxygenases and tyrosine in catalases (1, 2). Cytochrome-c peroxidase (CCP; EC 1.11.1.5) catalyzes the oxidation of cytochrome c (cyt c) by H202 through an intermediate state (ES) that consists of a ferryl (Fe4+=O) heme and a free radical localized on Trp-191 (3). The buried carboxylate of Asp-235 stabilizes this free radical and also accepts a hydrogen bond from the histidine heme ligand, His-175. The resulting Asp/His/metal triad is a common motif in metalloproteins and is reminiscent of the Asp/His/ Ser triad of serine proteases (4,5). In this study, we report the deletion of the histidine iron ligand of CCP to produce a heme enzyme which contains no covalent link to the protein. The facile occupation ofthis cavity by exogenous small molecules demonstrates the potential for producing heme catalysts containing a wide range of unnatural ligands.
MATERIALS AND METHODSProtein Expression and Purification. Wild-type and mutant CCP proteins were overexpressed in Escherichia coli BL21(DE3) using the plasmid pT7CCP (6). Wild-type CCP in our laboratory is that with Met-Lys-Thr at the N terminus and containing Ile-53 and Gly-152 (7). The His-175 --Gly (H175G) mutant was created by site-directed mutagenesis, expressed, purified to homogeneity, and reconstituted with heme as described (6). This purified reconstituted protein was crystallized twice against distilled water and stored as a crystal suspension at 77 K. Protein concentrations were determined from the extinction coefficients determined by pyridine hemochromogens (8, 9). In the case of H175G the extinction coefficient was determined for the 280-nm band because of the variability (with pH and temperature) in the intensity and energy of the Soret band (see below).X-Ray Crystallography.t Attempts to obtain crystals of H175G by ...
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