Cannons A C, Barber M J, Solomonson L P
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
J Biol Chem. 1993 Feb 15;268(5):3268-71.
A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.
一种对应于普通小球藻同化型NADH：硝酸还原酶假定血红素结合结构域的重组蛋白已在转化的大肠杆菌BL21细胞中表达并纯化。该重组蛋白的亚基分子量约为10 kDa，其N端序列以PAGA残基开始，与cDNA分析预测的一致。该蛋白的紫外可见光谱证实了血红素的掺入，氧化型和还原型的最大吸收峰分别在413 nm以及423、528和557 nm处。圆二色光谱表明血红素发色团的环境与天然酶非常相似。重组血红素结构域的电位滴定产生的中点电位为+16 mV（n = 1，pH 7），大大高于天然酶获得的-160 mV和先前表达的包含部分钼蝶呤结构域的重组血红素结构域获得的-28 mV的值。这些结果表明，参与小球藻硝酸还原酶钼蝶呤结构域形成的部分氨基酸序列影响血红素辅基的氧化还原电位。
