Transmembrane movement of heme

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.