Reduction potential and heme-pocket polarity in low potential cytochrome b5 of Giardia intestinalis.

Abstract Although it lacks mitochondria and the ability to synthesize heme, the protozoan parasite Giardia intestinalis encodes several heme proteins. This includes four members of the cytochrome b 5 family, three of which are of similar size to mammalian cytochromes b 5 but with reduction potentials that are 140 to 180 mV lower. While no structures have yet been determined for any of these proteins, homology modeling points to an increase in heme pocket polarity as a reason for their low potentials. To test this we measured the reduction potentials of four mutants of Giardia cytochrome b 5 isotype-I (gCYTB5-I) in which polar residues at two candidate positions (C84, Y51) in the heme pocket were changed to nonpolar ones (C84A, C84F; Y51L, Y51F). All mutants were expressed with comparable levels of heme incorporation and had UV–visible spectra consistent with low spin bis-histidyl coordination. These mutations increased the reduction potential by 18 to 57 mV and highlight the influence of C84, which is a residue unique to gCYTB5-I and whose mutation to alanine caused the largest increase. The influence of these two residues plus that of Y61 reported previously accounts for much of the reduction potential difference between gCYTB5-I and microsomal cytochrome b 5 . A complementary triple mutant of the latter with the hydrophilic residues found in gCYTB5-I bound heme less effectively but nonetheless had a reduction potential that was 135 mV lower than wild type.