5.B.1 The gp91phox Phagocyte NADPH Oxidase-associated Cytochrome b558 (Phox) Family
The human phagocyte cytochrome b558 is a heterodimeric complex consisting of a heavy (β) chain (gp91phox) and a light (α) chain (p22phox) as well as several auxiliary subunits (Geisz and Leto, 2004). The β-chain is a glycoprotein of 570 amino acyl residues called gp91phox, the product of the X-linked chronic granulomatous disease gene. The protein bears (1) the heme-binding site in its N-terminal 280 residues, and (2) an FAD binding site (residues 338-344) as part of the C-terminal NADPH oxidase domain. The N-terminal domain has 6 putative transmembrane spanners (TMSs) and is the cytochrome binding site. It has been reported to catalyze efflux of protons through an H+ channel that acts as a charge compensation pathway for the electrogenic generation of the superoxide radical, O2&149;-. The proposal that (gp91phox) has H+ channel activity has been effectively disputed and is now in doubt (DeCoursey 2003; DeCoursey et al. 2002, 2001, Morgan et al. 2002, 2007). Further, the voltage-gated proton channel (TC# 1.A.51) is expressed on phagosomes (Okochi et al., 2009 ). As a result, this family has been removed from class 1.A where it previously assumed the TC#1.A.20.
gp91phox is the terminal component of a respiratory chain that transfers single electrons from cytoplasmic NADPH to O2 on the external side of the plasma membrane generating superoxide. Its activity is electrogenic, causing depolarization of the membrane potential, negative inside. Four histidines, two in TMS3 (his-101 and his-115) and two in TMS5 (his-209 and his-222), are known to ligand heme (Biberstine-Kinkade et al. 2001). p22phox (the α-chain) is not homologous to anything else in the databases and may not have catalytic activity.
The activity of gp91phox is accompanied by a fall in the internal pH. Efflux of H+ through a channel would be expected to provide charge compensation, preventing a large fall in the internal pH. In the presence of arachidonic acid, the direction of H+ flux is dictated by the pmf. The oxidase interacts with various cytosolic proteins that may regulate its activity. Differential cell surface expression of Nox1, Nox2 and Nox5 (for example) is accomplished via two pathways, a Sar1 (small GTPase)-dependent pathway and a Sar-independent pathway (Kiyohara et al. 2018).
gp91phox is homologous throughout its length to proteins from animals, slime molds, plants, fungi and bacteria. There are seven homologues in humans (Nox1-5 and Duox1 and 2). Homologues in all organisms form six phylogenetic clusters (Kimball and Saier, 2002). These include mitogenic oxidases of animals, the so-called respiratory burst oxidase proteins of plants, and the ferric reductases of yeast and plants. In all cases, electrons are probably transferred across the membrane (from in to out) forming superoxide. Sixteen homologues of the Phox family have been sequenced from A. thaliana. Homologues are also found in C. elegans and D. melanogaster. Duox enzyme activities in epithelia are inhibited by compounds that block Hv1, but inhibition occurs through Hv1-independent mechanisms, supporting the idea that Hv1 is not required for Duox activity (Gattas et al. 2019).
The overall electron transfer reaction catalyzed by gp91phox and some of its homologues is:
1. electron (in) → electron (out)
2. O2 (out) + electron (in) → O2- (superoxide) (out)