Multi-Heme Self-Assembly in Phospholipid Vesicles

Abstract

The use of weak, intermolecular forces to orchestrate the contruction of multicomponent systems in membranes has significant implications in diverse areas of chemistry, biology, and medicine. We describe here the construction and characterization of multi-heme molecular ensembles in phospholipid vesicles. A trianionic zinc porphyrin was designed to bind cytochrome c at the membrane surface, while being anchored to a membrane spanning manganese porphyrin in the membrane interior via a terminal imidazole. The structure of the construct was probed by fluorescence and UV spectroscopy. Cytochrome c formed a stoichiometric 1:1 complex with the anionic porphyrin with a high binding constant (K a ≈ 5 × 106 M-1). The ligation of the imidazole to the manganese porphyrin was confirmed by UV spectral changes. Large differences in the fluorescence quenching of Zn porphyrins with and without the terminal imidazole were observed upon their insertion into vesicles containing the Mn porphyrin. These spectroscopic observations were consistent with the formation of a ligated, ternary system consisting of the Mn(II) porphyrin, the imidazole-tailed zinc porphyrin acting as a bridge, and the surface associated cytochrome c. The nature of the binding of cytochrome c at the membrane−water interface was investigated by Langmuir−Blodgett (LB) and differential scanning calorimetric (DSC) techniques. The data obtained suggested that the protein was surface bound with minimal penetration into the membrane. LB studies were also used to probe the orientation of the trianionic porphyrin moiety at the membrane surface, and an edge-on orientation was inferred from the data. The formation of a stable vesicular system was confirmed by the formation of well-defined DSC thermograms. Phase separation was observed at high porphyrin:lipid ratios. Electron transfer from the Mn(II) in the membrane interior to the surface bound ferricytochrome c was investigated, as a probe both for spatial definition of the ensemble and for the elucidation of electron transfer mechanism in the genre of weakly coupled systems over large distances. Trianionic Zn porphyrins with varying tether lengths (12, 8, and 4 carbons) were used. The electron transfer rate was found to be first order and independent of the tether length, indicative of medium mediated electron transfer via multiple pathways. Comparison to similar systems in the literature yielded a predicted distance of ∼23 Å between the Mn and Fe centers in DMPC/DPPC vesicles. This distance suggested that the protein was surface bound to the membrane and separated from the Mn porphyrin by the thickness of one leaflet of the phospholipid bilayer. In thinner DLPC vesicles the predicted increase in the electron transfer rate was observed. Additionally, electron transfer was observed to be bimolecular in systems where trianionic porphyrins lacking the imidazole tether were used to recruit the cytochrome c.

Electron transfer kinetics and morphology of cytochrome c at the biomimetic phospholipid layers

“…The possible reason is that the redox center of cyt c easily penetrated into the shorter chain length of lipids. The same tendency was observed in the previous work [32] studied for the electron transfer in vesicles composed of lipid bilayer. It showed that the electron transfer rate between the redox centers decreased as the chain length of lipids increased due to a low driving force at large distances.…”

Supramolecular Complex of Cytochrome c with a Polyanionic β‐Cyclodextrin

“…Uroporphyrin I with eight carboxylate groups caused formation of a ground‐state complex with cyt c leading to static fluorescence quenching of uroporphyrin I by cyt c 6. Groves and co‐workers constructed a sophisticated multiporphyrin assembly by partly using electrostatic interactions between an anionic zinc porphyrin and cyt c 7. In 1997, Hamilton and co‐workers reported a well‐designed protein receptor composed of a calix[4]arene building block and four peptide loops with eight carboxylate groups 8.…”

Fatty acids, fatty acid analogues and their derivatives

“…353 The construction and characterization of multi-heme ensembles in phospholipid vesicles have been described. 354 Aggregation of stilbene derivatized fatty acids and phospholipids in monolayers and vesicles has been studied. 355 A number of bis-cationic dimeric lipids have been synthesized and the vesicles formed show widely different membrane organizations with exceptional thermotropic properties.…”