Metabolic Pathways of Bacterial Nitrification 401 



or a Gary spetrophotometer, found that intact Nitwbacter cells 

 or various cell-free fractions possessing nitrite oxidase activity 

 showed upon addition of nitrite, absorption maxima in the 550 

 and 520 m/' regions (representative of the alpha and beta peaks 

 of a cytochrome component of the c type) and in the 585-590 

 mu and 438 m/x regions (indicative of the alpha and gamma peaks 

 of a cytochrome a type, probably «i) (Fig. 3). The cell-free 

 fraction, 144,000 x g supernatant wliicli lacked nitrite oxidase 

 activity gave smaller absorption maxima at 550 m,a and negligible 

 peak at 585-590 m,". The results suggested that nitrite oxidation 

 cannot take place if cytochrome oxidase (cyt. ai-like component) 

 is absent and implicated both cytochrome c- ( 551 ) and a- ( 589 ) - 

 like components in the action of nitrite oxidase. Addition of 

 FeSOi to the particulate enzyme inactivated by cyanide dialysis 

 resulted in the appearance of reduced cytochrome peaks, probably 

 due to the non-enzymatic reduction of cytochromes by iron. 

 Ferric ions in the absence or presence of nitrite, oddly enough, 

 failed to restore the steady state absorption spectra of the cyanide 

 inactivated nitrite oxidase. These observations in general strongly 

 suggest that biological nitrite oxidation occurs in a series of 

 enzymatic steps involving the sequential transfer of electrons 

 from nitrite to molecular oxygen via cytochromes c- and fli-like 

 components (4). It may be that iron is acting between NOr and 

 cytochrome c but the exact role of the metal is not yet clear. 



Cell-free extracts of Nitrobacter possess an active NO."cyto- 

 chrome c reductase* which catalyzes the transfer of electrons 

 from nitrite to mammalian cytochrome c. Presumably its natural 

 electron acceptor is the cytochrome c-like component of Nitro- 

 bacter. The further transport of electrons from cytochrome c 

 to Oi- is catab'zed by the cytochrome oxidase present in the 

 extracts.* The pathway of electron transport involved in nitrite 

 oxidation may be represented by the following scheme: 



Nitrite-cytochrome c ^ i . ^ 



■i' ~ Cytochrome oxidase 



reductase ^ 



NO2" ► cytochrome c ► cyt. a, ► O2 



unpublished data. 



