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PMID: 1320378 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

The Biochemical journal ·Vol. 284 ( Pt 3) ·1992-06-15 ·Pages 781-8

Segal AW, West I, Wientjes F, Nugent JH, Chavan AJ, Haley B, Garcia RC, Rosen H, Scrace G

Abstract

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.

MeSH Terms
Amino Acid Sequence Animals Binding Sites Cell Line Cell Membrane/metabolism Chromatography, Affinity Chromatography, Ion Exchange Cytochrome b Group/chemistry,isolation & purification,metabolism Electron Spin Resonance Spectroscopy Flavin-Adenine Dinucleotide/analysis,metabolism Heme/analysis,metabolism Humans Macromolecular Substances Models, Structural Molecular Sequence Data Molecular Weight NADH, NADPH Oxidoreductases/blood NADP/metabolism NADPH Oxidases Neutrophils/enzymology Oxidation-Reduction Phagocytosis Protein Conformation Rats Sequence Homology, Nucleic Acid Superoxides/metabolism
Chemicals
Cytochrome b Group Macromolecular Substances cytochrome b245 Superoxides Flavin-Adenine Dinucleotide Heme NADP NADH, NADPH Oxidoreductases NADPH Oxidases
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Segal A W
Department of Medicine, University College London, Rayne Institute, U.K.
West I
Wientjes F
Nugent J H
Chavan A J
Haley B
Garcia R C
Rosen H
Scrace G
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2840781
Published
1992-06-15
Pages
781-8
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1132607
Subset
IM
Grants
NIAID NIH HHS · AI25606 · United States
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1042/bj2840781 PMC Full Text View journal details
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