Structural and functional analyses of membrane-spanning electron transfer system in neuroendocrine secretory vesicles by utilizing proteoliposome

1. 细胞色素b561细胞色素b561存在于中枢和周围神经系统的神经内分泌囊膜中。它起着跨膜电子通道的作用；即在囊泡表面从抗坏血酸接受一个电子当量，并在囊泡表面提供一个电子当量的单脱氢抗坏血酸自由基。先前我们通过EPR光谱和血红素含量分析发现细胞色素b561含有两个独立的血红素中心（gz = 3.12和gz = 3.69种）。氧化还原电位测量天然细胞色素b561在洗涤剂溶解状态下的氧化还原电位测量显示存在两种具有不同中点电位（+155和+62 mV）的氧化还原组分。氧化状态下的细胞色素b561经DEPC处理后，低氧化还原组分急剧降低，低至-30 mV。这一观察结果与DEPC处理通过细胞色素b561.3的保守残基（His88， His161和Lys85）的特异性n -碳氧基化导致asco…More rate的电子接受能力受到抑制的观点一致。EPR谱研究了depc处理对细胞色素b561氧化后EPR谱的影响。然而，在原生和经depc处理的样品之间没有明显的差异。在天然样品中加入抗坏血酸后，两个血红素中心可以完全还原。而对于经depc处理的样品，只有gz = 3.12信号消失。gz = 3.69的信号仍保持全强度。这一结果表明，gz = 3.69种具有在囊外接受抗坏血酸电子的作用，并且易受depc修饰的影响。细胞色素b561支持的跨膜电子转移纯化的细胞色素b561成功地在内向外的方向上重组成负载抗坏血酸的囊泡膜。细胞色素b561可以通过跨膜电子转移向胞外细胞色素c提供等量的电子。此外，在没有任何氧化还原介质的情况下，细胞色素b561可以支持泡外多巴胺b-羟化酶的单加氧酶活性。添加铁氰化物作为氧化还原介质，提高了囊外单加氧酶的活性。少

1. Cytochrome b561Cytochrome b561 exists in the membranes of neuroendocrine vesicles of central and peripheral nervous systems. It functions as a transmembrane electron conduit ; I. e. receiving one electron equivalent from ascorbate at the intravesicular surface and donating one electron equivalent to monodehydro ascorbate radical at the intravesicular surface. We found previously that cytochrome b561 contained two independent heme centers (gz = 3.12 and gz = 3.69 species) by EPR spectroscopy and heme content analysis.2. Redox Potential MeasurementRedox potential measurements of native cytochrome b561 in a detergent-solubilized state showed a presence of two redox components with a different midpoint potential (+155 and +62 mV). DEPC treatment of cytochrome b561 in the oxidized state caused a drastic lowering of the lower redox component as low as -30 mV. This observation is consistent with the notion that the DEPC treatment caused an inhibition of electron accepting ability from asco … More rbate by the specific N-carbethoxylations of conserved residues (His88, His161 and Lys85) of cytochorme b561.3. EPR SpectroscopyEffect of the DEPC-treatment on the oxidized EPR spectrum of cytochrome b561 was examined. However, there was no appreciable difference between the native and the DEPC-treated samples. Upon addition of ascorbate to the native sample, both heme centers could be fully reduced. However, for the DEPC-treated sample, only the gz = 3.12 signal disappeared. The gz = 3.69 signal remained in full intensity. This result suggests that the gz = 3.69 species has a role for the electron accepting from ascorbate at the extravesicular site and is susceptible to the DEPC-modification.4. Transmembrane Electron Transfer Supported by Cytochrome b561Purified cytochrome b561 was successfully reconstituted into ascorbate-loaded vesicle membranes in an inside-out orientation. Cytochrome b561 could donate electron equivalent to extravesicular cytochrome c via transmembrane elecron transfer. Further, cytochrome b561 could support the monooxygenase activity of extravesicular dopamine b-hydroxylase in the absence of any redox mediator. Addition of ferricyanide as a redox mediator enhance the extravesicular monooxygenase acitivity. Less