House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding

Abstract

We report that cytochrome b 5 (cyt b 5) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b 5 that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180° rotation about the α-γ-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.2:1 ratio of rotational forms in freshly expressed protein. The equilibrium ratio (5.5:1) is established only upon incubation at temperatures above 37 °C. House fly Mc cyt b 5 is only the second b-hemoprotein that has been shown to exhibit kinetically trapped hemin at room temperature or above, the first being cyt b 5 from the outer membrane of rat liver mitochondria (rat OM cyt b 5). Finally, we show that the small excess of one orientational isomer over the other in freshly expressed protein results from selective binding of hemin by the apoprotein, a phenomenon that has not heretofore been established for any apocyt b 5.