Comparison of cytochromes b₅ from insects and vertebrates

Abstract

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

Molecular modeling of cytochrome b 5 with a single cytochrome c-like thioether linkage

“…There is therefore little chance that these two cysteine residues form a disulfide bond. On the other hand, Cys88 likely functions as an α6 C cap in DS cyt b 5 , a role assigned to Cys84 in frog OM cyt b 5 species [19]. A previous study also found that Cys, a nonpolar residue, is the fifth most overrepresented residue at the C cap position in short 3 10 helices [49].…”

“…3a). Meanwhile, in the X-ray structures of bovine and HF cyt b 5 , the 3 10 helix observed is in the Cterminus, and is defined as α6 [1,19]. On the other hand, several 3 10 helices were transitionally formed in the simulation of apo-cyt b 5 as well as its variants, and these were observed in the regions adjacent to the C-terminus of α2 and α5 [30], just as in DS cyt b 5 .…”

“…To date, only one cyt b 5 from an insect-house fly cyt b 5 (HF cyt b 5 )-has been characterized structurally, by Benson and co-workers [19]. For other insect cyt b 5 , such as domestic silkworm cyt b 5 (DS cyt b 5 ), the amino acid sequence was only recently identified (GenBank ABF51280.1).…”

Functional and structural characterisation of a viral cytochrome b5

“…Data were processed using MOSFLM through the XIA2 pipeline [23,24]. The structure was solved by molecular replacement (MOLREP, [25]) using the cytochrome b5 from Musca domestica as a model (PDB code 2IBJ, [26]) which shares 35% amino acid identity.…”

Whole-genome duplications contributed to the expansion of cytochrome b5 genes in Paramecium tetraurelia

“…Cyt b 5 is found as a membrane component or as a soluble form in cells. Its heme-binding region consists of 6 α-helices and 5 β-strands (β1-α1-β4-β3-α2-α3-β5-α4-α5-β2-α6) (Wang et al, 2007). As an electron transfer component, cyt b 5 is involved in a number of oxidative reactions, including the anabolic metabolism of fats and steroids, as well as the catabolism of xenobiotics and endogenous metabolism compounds (Schenkman and Jansson, 2003).…”