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 approximate to 5 x 10(6) 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 similar to 23 Angstrom 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.