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

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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.