A structural model for the nucleotide binding domains of the flavocytochrome b_–245 β‐chain

Abstract

NADPH is a system in phagocytic cells that generates 02-and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the / 3 subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b"245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.

Functional Epitope on Human Neutrophil Flavocytochrome b558

“…This region of the molecule has been identified as a potential p47 phox contact site based on CGD analysis (46), synthetic peptide inhibition of oxidase activation (43,45) and translocation (44), and selection by phage display analysis of p47 phox affinity matrices (45). Such a placement also suggests that the C-terminal residues of gp91 phox , 566 NKENF 570 (not shown in the model), might lay over the cleft proposed to contain the NADPH binding site of gp91 phox and would allow Phe 570 to coordinate the isoalloxazine ring of the FAD as originally suggested by Taylor et al (47) from studies of similarity to the ferridoxin nucleotide reductases. According to this model, 555 ESGPRGVH-FIF 565 is well-removed from the NL7 epitope (shown in green), having at least 15Å separating the two regions at closest approach.…”

Cloning and sequencing of the bovine flavocytochrome b subunit proteins, gp91-phox and p22-phox: comparison with other known flavocytochrome b sequences

“…However, because of the complex nature of this heterodimeric membrane protein, the specific functional and structural features of flavocytochrome b have not been fully characterized. Thus what little is known about the important functional and structural sites of these two proteins has come mainly from natural genetic mutations of these proteins [32], peptide mapping studies [33][34][35], and comparison of areas of conservation between the amino acid sequences obtained from other flavoproteins [28,29,36]. However, flavocytochrome b homologs have been sequenced from only a few species other than human.…”

Functional analysis of two-amino acid substitutions in gp91phox in a patient with X-linked flavocytochrome b558-positive chronic granulomatous disease by means of transgenic PLB-985 cells

“…With the program SwissPDB Viewer, a picture of the three-dimensional (3D) model of gp91phox was made and the residues His303 and Pro304 were highlighted (Taylor et al 1993;Guex and Peitsch 1997).…”

“…The functional significance of the His303Asn and Pro304Arg mutation was further analysed in a 3D-model of the C-terminal tail of gp91phox (Taylor et al 1993;Leusen et al 2000). This model shows that both His303 and Pro304 are located at the surface of the protein, at the N-terminal of the beta-sheet (βF2) in hydrophilic surroundings (Fig.…”