Roma Glenn W, Crowley Louis J, Barber Michael J
Department of Molecular Medicine, School of Basic Biomedical Sciences, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd, MDC 007, Tampa, FL 33612, USA.
Arch Biochem Biophys. 2006 Aug 1;452(1):69-82. doi: 10.1016/j.abb.2006.04.021. Epub 2006 May 24.
Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.
细胞色素b5还原酶（cb5r）是氧化还原酶中黄素蛋白转氢酶家族的成员，催化还原当量从生理电子供体NADH转移至两分子细胞色素b5。我们已确定了犬（Canis familiaris）推定的全长膜相关酶的正确核苷酸序列，并构建了一个异源表达系统，用于生产可溶性催化黄递酶结构域的组氨酸标签变体，该结构域包含第33位至第300位氨基酸残基。通过简单的两步色谱法，重组黄递酶结构域已被纯化至同质，并被证明是一种分子量为31,364（m/z）的简单黄素蛋白，保留了NADH：铁氰化物还原酶和NADH：细胞色素b5还原酶活性。重组蛋白含有完整的FAD，并表现出与使用相同表达系统产生的大鼠细胞色素b5还原酶黄递酶结构域的重组形式相当的吸收光谱和圆二色光谱，表明蛋白质折叠相似。氧化还原电位滴定得出FAD/FADH2电对的标准中点电位（Eo'）为-273±5 mV，这与相应大鼠结构域获得的值相同。热变性研究表明，犬结构域表现出与大鼠蛋白相当的稳定性，证实了相似的蛋白质构象。初始速率动力学研究表明，犬黄递酶结构域对NADH作为还原底物的偏好明显高于NADPH，并且对于NADH：铁氰化物还原酶和NADH：细胞色素b5还原酶活性，其催化常数（kcat）分别为767和600 s-1，对NADH、铁氰化钾（K3Fe(CN)6）和细胞色素b5的米氏常数（Km）分别为7、8和12 μM。针对多种NAD+类似物测定的光谱结合常数（Ks）表明，对APAD+（Ks = 71 μM）和PCA+（Ks => 31 mM）分别观察到最高和最低亲和力，并表明了吡啶核苷酸各部分的结合贡献。这些结果提供了犬cb5r全长膜相关形式的首个正确序列，并使用相同表达系统对来自两个系统发育来源的酶进行了直接比较，表明这两种酶具有可比的光谱、动力学、热力学和结构特性。
