Biochemistry, University of KwaZulu-Natal, Pietermaritzburg, 3201, South Africa.
Malar J. 2022 Jun 7;21(1):173. doi: 10.1186/s12936-022-04188-5.
Copper is an essential metal for living organisms as a catalytic co-factor for important enzymes, like cytochrome c oxidase the final enzyme in the electron transport chain. Plasmodium falciparum parasites in infected red blood cells are killed by excess copper and development in erythrocytes is inhibited by copper chelators. Cytochrome c oxidase in yeast obtains copper for the Cu site in the Cox1 subunit from Cox11.
A 162 amino acid carboxy-terminal domain of the P. falciparum Cox11 ortholog (PfCox11Ct) was recombinantly expressed and the rMBPPfCox11Ct affinity purified. Copper binding was measured in vitro and in Escherichia coli host cells. Site directed mutagenesis was used to identify key copper binding cysteines. Antibodies confirmed the expression of the native protein.
rMBPPfCox11Ct was expressed as a 62 kDa protein fused with the maltose binding protein and affinity purified. rMBPPfCox11Ct bound copper measured by: a bicinchoninic acid release assay; atomic absorption spectroscopy; a bacterial host growth inhibition assay; ascorbate oxidation inhibition and in a thermal shift assay. The cysteine 157 amino acid was shown to be important for in vitro copper binding by PfCox11whilst Cys 60 was not. The native protein was detected by antibodies against rMBPPfCox11Ct.
Plasmodium spp. express the PfCox11 protein which shares structural features and copper binding motifs with Cox11 from other species. PfCox11 binds copper and is, therefore, predicted to transfer copper to the Cu site of Plasmodium cytochrome c oxidase. Characterization of Plasmodium spp. proteins involved in copper metabolism will help sceintists understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.
铜作为细胞色素 c 氧化酶（电子传递链中的最后一种酶）等重要酶的催化辅助因子，是生物体必需的金属。感染红细胞中的疟原虫寄生虫被过量的铜杀死，铜螯合剂抑制红细胞发育。酵母中的细胞色素 c 氧化酶从 Cox11 亚基中的 Cox1 获得铜位点的铜。
重组表达了疟原虫 Cox11 同源物（PfCox11Ct）的 162 个氨基酸羧基末端结构域，并对 rMBPPfCox11Ct 进行了亲和纯化。在体外和大肠杆菌宿主细胞中测量了铜结合。使用定点突变鉴定关键的铜结合半胱氨酸。抗体证实了天然蛋白的表达。
rMBPPfCox11Ct 表达为与麦芽糖结合蛋白融合的 62 kDa 蛋白，并进行了亲和纯化。rMBPPfCox11Ct 通过以下方法结合铜：双缩脲酸释放测定；原子吸收光谱法；细菌宿主生长抑制测定；抗坏血酸氧化抑制和热转移测定。结果表明 PfCox11 的第 157 个氨基酸残基对体外铜结合很重要，而 Cys60 则不重要。针对 rMBPPfCox11Ct 的抗体检测到天然蛋白。
疟原虫属表达 PfCox11 蛋白，该蛋白与其他物种的 Cox11 具有结构特征和铜结合基序。PfCox11 结合铜，因此预测将铜转移到疟原虫细胞色素 c 氧化酶的 Cu 位。对参与铜代谢的疟原虫属蛋白的特性研究将有助于科学家了解细胞色素 c 氧化酶和这种必需金属在疟原虫体内平衡中的作用。
