Segal A W, West I, Wientjes F, Nugent J H, Chavan A J, Haley B, Garcia R C, Rosen H, Scrace G
Department of Medicine, University College London, Rayne Institute, U.K.
Biochem J. 1992 Jun 15;284 ( Pt 3)(Pt 3):781-8. doi: 10.1042/bj2840781.
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.
吞噬细胞的NADPH氧化酶对于有效杀灭和消化摄入的微生物至关重要。一种非常特殊的低电位细胞色素b（b-245）是该系统中唯一已被鉴定的氧化还原分子。结合NADPH并将电子传递给细胞色素的含FAD黄素蛋白三十年来一直未被鉴定出来。我们在此表明，该氧化酶激活后膜中的血红素/FAD比值没有显著变化，这表明FAD从一开始就存在于膜中，而非从胞质溶胶中募集而来。缺乏细胞色素b的X连锁慢性肉芽肿病（CGD）患者细胞的膜中FAD含量约为正常受试者以及缺乏胞质蛋白p47-phox的常染色体隐性CGD患者的四分之一。未诱导的早幼粒细胞（HL60）细胞中也存在类似低量的FAD，这表明X-CGD患者细胞中FAD含量低可能与该氧化酶系统无关。细胞色素b-245似乎以2:1的摩尔比结合血红素和FAD。纯化的细胞色素的电子顺磁共振信号较弱，在g = 3.31处有一个不对称的g(z)峰。在有脂质存在的情况下，纯化的细胞色素可部分重新黄素化（约20%）。在该细胞色素b的β亚基与假定的NADPH和FAD结合位点的铁氧化还原蛋白-NADP+还原酶（FNR）还原酶家族之间检测到氨基酸序列同源性。32P标记的2-叠氮基-NADP被用作NADPH结合位点的光亲和标记。在细胞色素的β亚基区域观察到与NADP竞争的标记。在三名该细胞色素缺失的X-CGD患者以及一名该细胞色素存在但在假定的NADPH结合位点有脯氨酸-组氨酸换位的患者中，该区域未观察到标记。这些研究表明细胞色素b-245是一种黄素细胞色素，是高等真核细胞中首次描述的，具有NADPH氧化酶完整的电子传输装置。
