398 



F. Egami, M. Ishtmoto and S. Taniguchi 



observed (Table 6). Analysis of flavin in the homogeneous preparation, as 

 recorded in Table 7, led to the conclusion that the flavin content in the 

 enzyme is close to the limit of error of determination. So the flavin content 

 is, if any, only a trace and the enzyme may be regarded as a metalloprotein 



O.D. 



0.2 



Oxidized] 

 -[Reduced] 



NaBH4 + KN03(IOpmoles) 



400 500 600 rn^L 



Fig. 2. Absorption spectrum of purified nitrate reductase. The solution contained 



NaR preparation (specific activity: 15 x 10* units/mg N) in final concentration 



of 1-9 mg protein/ml in phosphate buffer 0-05 m, pH 7-1 in Thunberg tube-type 



cuvette at 30°C. 



but not as a metalloflavoprotein. In our previous preUminary communica- 

 tion (Taniguchi and Itagaki, 1959), it was considered to be a metalloflavo- 

 protein. 



Purified solubiUzed nitrate reductase does not reduce nitrate w^ith formate 

 or DPNH, but reduces nitrate with MVH and, less actively, with reduced 

 flavin adenine dinucleotide (FAD), riboflavin phosphate (FMN) and free 

 riboflavin (Table 8). CN" and Ng^ strongly inhibit the reaction (Table 9). 



CO (1 atni in the dark) has no effect. 



Nitrate reductase thus obtained may be regarded as a solubiUzed form of 

 the terminal oxidoreductase in the particulate fraction and so is an anaerobic 

 variant of terminal oxidases (Fig. 3). 



HOQNO 



Formate 



(FAD)— t-cyf b, 



Incubation in cold 

 after 5 min heating 

 of eCC.pH 8.3. 



-«-NaR 



t 

 FADH?, MVH 



no; 



phenosofranine - Hg 

 Fiu. 3. Particulate nitrate-reducing system. 



