González C, Brito N, Marzluf G A
Department of Biochemistry, Ohio State University, Columbus 43210, USA.
Mol Gen Genet. 1995 Dec 10;249(4):456-64. doi: 10.1007/BF00287108.
Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.
粗糙脉孢菌的硝酸还原酶是一种复杂的多氧化还原蛋白，由两个相同的亚基组成，每个亚基包含三个不同的结构域，一个氨基末端结构域，其中含有一个钼蝶呤辅因子；一个中央含血红素结构域；以及一个羧基末端结构域，该结构域结合一个黄素和一个吡啶核苷酸辅因子。硝酸还原酶的黄素结构域似乎与铁氧化还原蛋白NADPH还原酶（FNR）具有结构和功能上的相似性。以FNR的晶体结构和众多硝酸还原酶中的氨基酸同一性为指导，采用定点诱变来替换怀疑参与黄素或吡啶核苷酸辅因子结合且因此对黄素结构域的催化功能很重要的特定氨基酸。每个突变的黄素结构域蛋白在大肠杆菌中表达，并分析其NADPH：铁氰化物还原酶活性。通过将每个操纵的基因转化到粗糙脉孢菌的nit-3无效突变体中，还研究了每个氨基酸取代对完整硝酸还原酶反应活性的影响。我们的结果确定了对硝酸还原酶黄素结构域功能至关重要且似乎对黄素或吡啶核苷酸辅因子的结合很重要的氨基酸残基。
