The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.

Functionalized double-chain surfactant 1 (potassium O,O ‘-didodecylphosphorodithioate) was synthesized. Its small unilamellar vesicles were characterized by dynamic laser light scattering and differential scanning calorimetry and its giant vesicles by phase-contrast optical microscopy. Also, 1's giant vesicles containing fluorescent dye 4a (5-carboxyfluorescein) or 4b [5-(dodecanamido)fluorescein] were characterized by epifluorescence microscopy. In a pH 9.0 borate buffer at 25 °C, vesicular 1 reacted with 2 (2-chloroethyl phenyl sulfide), a simulant for the chemical warfare agent mustard [bis(2-chloroethyl) sulfide], to give 5 [S-[(2-phenylthio)ethyl] O,O‘-didodecylphosphorodithioate], involving capture of reactive intermediate cation 9 (1-phenylthiocyclopropane) by the anion of 1. This reaction was accompanied by the precipitation of 5, which resulted in wounding/destruction of the vesicles and the release of dye 4a (from giant vesicles). The combination of the conversion of 2 into 5 and dye release suggests the potential of vesicular systems for simultaneous decontamination and signaling of chemical agents. 2 hydrolyzed to give only 6 [2-(phenylthio)ethanol] in the pH 9.0 buffer at 25 °C.

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