Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket.

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The stability of the heme-globin linkage in ␣␤ dimers and in the isolated chains of human hemoglobin has been probed by studying the transfer of heme from the proteins immobilized onto CNBr-activated Sepharose 4B to human albumin. The kinetic and equilibrium features of the reaction have been measured spectrophotometrically given the stability of the heme donors and the ease with which heme donor and acceptor can be separated. Isolated ␣ and ␤ chains transfer heme to albumin at similar rates (1-6 ؋ 10 ؊2 s ؊1 at pH 9.0 and 20°C) in the ferrous CO-bound and in the ferric state. In ␣␤ dimers the heme-globin linkage is strengthened considerably, albeit to a different extent in the ferrous CO-bound and ferric met-aquo derivatives. Only in the latter heme is lost at a measurable rate, 0.065 ؎ 0.011 ؋ 10 ؊2 s ؊1 for ␣ heme and 2.8 ؎ 0.6 ؋ 10 ؊2 s ؊1 for ␤ heme at pH 9.0 and 20°C, which is very close to the rate measured with soluble met-aquo-hemoglobin at micromolar concentrations. These results indicate that in human hemoglobin the heme-globin linkage in the ␣ chains is stabilized by interactions between unlike chains at the ␣ 1 ␤ 1 interface, whereas heme binding to the ␤ chains is stabilized by interactions at the ␣ 1 ␤ 2 interface. These long range factors have to be taken into account in addition to the local factors at the heme pocket when evaluating the effect of point mutation and chemical modification.

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Apo-myoglobin covalently linked on CNBr-activated Sepharose 4B is proposed as a new heme acceptor for investigating the heme transfer reaction from hemoproteins. Immobilized apo-myoglobin has the desirable properties of an ideal heme acceptor in that it is characterized by a high affinity for ferric heme, a high stability towards denaturation even at physiological temperatures and can be lyophilized for long-term storage. The study of heme release from myoglobin at pH 5.0 and 37³C indicates that heme affinity is increased at least 10-fold relative to the soluble protein. Experiments with human hemoglobin allowed the estimation of the heme release rates from both K K and L L chains and brought out the greater temperature sensitivity of the K K chain heme-globin linkage. z 1998 Federation of European Biochemical Societies.