Bioinorganic Chemical Study on Structure and Function of Cu-containing Nitrite Reductase

本课题的目的是阐明产铜嗜酸杆菌IAM1013、产碱杆菌和木糖嗜酸杆菌Gifu菌株含铜亚硝酸盐还原酶(NIR)的结构和功能1)产铜菌IAM1013、产碱杆菌和木糖嗜酸杆菌GIFU1051的含铜亚硝酸还原酶含有两种类型的铜(1型铜和2型铜)。用化学方法制备了2型贫铜(T2D)近红外光谱材料。目前的T2D酶不含2型铜。2)通过自然和二维近红外光谱的比较，确定了300-800 nm范围内的第二类铜位对光谱的贡献很小。3)根据近红外光谱分析系统，T2D-NIR显示了酶的活性，但比天然酶的活性低。4)在pH值为7.0时，分子内电子转移(ET)过程服从一级动力学，测得的速率常数为1,400-1,900s；-1&gt；(t_&lt；1/2&gt；=0.5msec)。在亚硝酸盐存在下，ET过程也遵循一级动力学，在相同pH下，速率常数减小1/12-1/2倍。亚硝酸盐会通过配位作用使2型铜的氧化还原电势发生变化，从而降低分子内的ET速率常数。5)4株岐阜菌(岐阜543、岐阜1048、岐阜1055、岐阜1764)的近红外光谱与产碱菌和GIFU1051菌株的近红外光谱非常相似。这些蓝色近红外光谱在594 nm处有最大吸收峰。6)Gifu 543和Gifu 1048菌株含有两种天青素(azurin-1和azurin-2)以及产碱杆菌和GIFU1051菌株，而Gifu 1055和Gifu 1764菌株只产生天青素-1。

The purpose of this project is to clarify the structure and function of Cu-containing nitrite reductases (NIRs) from Achromobacter cycloclastes IAM1013, Alcaligenes sp., and Achromobacter xylosoxidans GIFU strains.1) NIRs from Achromobacter cycloclastes IAM1013, Alcaligenes sp., and Achromobacter xylosoxidans GIFU1051 contain two types of Cu (type 1 Cu and type 2 Cu). Type 2 Cu-depleted (T2D) NIRs were prepared by a chemical treatment. The present T2D enzymes have no type 2 Cu. 2) The spectroscopic contribution of the type 2 Cu site in the region of 300-800 nm is ascertained to be quite small by a comparison between native and T2D NIRs. 3) According to the NIR assay system, T2D NIRs show the enzymatic activities, although they are lower than those of the native enzymes. 4) The intramolecular electron transfer (ET) process obeys first-order kinetics and observed rate constants are 1,400-1,900s^<-1> (t_<1/2>=ca.0.5 msec) at pH7.0. In the presence of nitrite, the ET process also obeys first-order kinetics with rate constants decreased by factors of 1/12-1/2 at the same pH.Nitrite would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased. 5) NIRs from four GIFU strains (GIFU 543, GIFU 1048, GIFU 1055, and GIFU 1764) are quite similar to those from Alcaligenes sp.and GIFU1051. These blue NIRs show the absorption maxima at 594 nm. 6) The strains of GIFU 543 and GIFU 1048 contain two kinds of azurin (azurin-1 and azurin-2) as well as Alcaligenes sp.and GIFU1051 strains, whereas the strains of GIFU 1055 and GIFU 1764 produce azurin-1 only.