Design Challenges for Hemoproteins:  The Solution Structure of Apocytochrome b₅^,

Abstract

In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

“…NMR studies suggest that while the membrane-anchoring regions of the apo-and holo-forms of cytochrome b 5 are structurally indistinguishable, the globular haem-binding domain of the apo-form is significantly more structurally disordered relative to the holo-form (Falzone et al 1996). Lee-Robichaud et al (1997) investigated the importance of the membrane-anchoring domain in the modulation of CYP17 lyase activity.…”

A test of the relationship between sequence and structure in proteins: Excision of the heme binding site in apocytochrome b5

“…The holoprotein has normal stability for a protein of this size and undergoes two-state thermal unfolding (Table 1 ; Pfeil, 1993). The low-resolution structure of the rat protein in the absence of the heme group (apocyt b,) has been obtained by NMR methods (Falzone et al, 1996). The / 3 topology is similar in the apo-and holoprotein forms but the absence of the heme loosens the P5 strand from the sheet and alters the helices enclosing the heme (Figs.…”

“…The / 3 topology is similar in the apo-and holoprotein forms but the absence of the heme loosens the P5 strand from the sheet and alters the helices enclosing the heme (Figs. IB, 2B) (Moore & Lecomte, 1993;Falzone et al, 1996). The structured segments define a part of the molecule that will eventually make few contacts with the prosthetic group.…”

“…The two-state unfolding and the observation that the ordered part folds autonomously (Moore & Lecomte, 1993) led to the proposal that apocyt b5 is made of two "modules," one for stability and pre-organization of the second, destined to bind the heme (Falzone et al, 1996). This hypothesis can be tested in various ways.…”

“…A first level of inquiry concerns the ability of the organizing module to adopt its fold independently of the binding module. In this work, data are reported on an abridged form of the rat apo- (Mathews et al, 1979) and (B) apocyt b5 (Falzone et al, 1996). The heme binding site is formed by the stretch joining p3 to p2, which contains in succession a2, a3, p5, a4, and a5.…”