TERMINAL H ELECTRON TRANSPORT SYSTEM 23 



DPN Neurospora 



Dehydrogenase — ^Ftavoprotein ^ Mo — ^Nitrate ^ NO3 



TPN I Reductase 



T Pseudomonas 



Factor — cyt. c — ^ .-NOj. NjO, Nj 



I Nitrite reductase 



\ E. coil 



Succinate ^-Succinic ■ »-cyt. b »- »-N03 



Dehydrogenase Nitrate reductase 



Fig. 9. Nitrate and nitrite as hydrogen acceptors. 



deiiitrificans, the reduction of nitrate may proceed in a stepwise manner to the production 

 of N2 and N2O ; in other instances, as in the case of B. licheniforms, B. subtihis or E. coli, 

 much of the nitrate may be reduced to NH3 (Delwiche, 1956). These alternatives are 

 depicted in Fig. 8. 



Pyridine nucleotides, flavoproteins, and cytochrome pigments participate in hydrogen 

 transport to nitrate as well as in hydrogen transport to oxygen. The hydrogen transport 

 system of Neurospora and E. coli which terminates in nitrate reduction is shown in Fig. 9. 



Fig. 9 also depicts a similar system for the reduction of nitrite in Pseudomonas (Ver- 

 hoeven, 1956). 



Enzyme preparations capable of reducing nitrate have been purified from E. coli, 

 Pseudomonas, fungi, and soy bean leaves. Reduction of nitrate by E. coli occurs in the 

 presence of TPNH2 or DPNH2 (Wainwright, 1955). FMN or FAD is required for maximal 

 activity. Menadione at a concentration of 10'^ M causes appreciable activation while Fe^^ 

 (A//80) still further activates when preincubated with the enzyme prior to addition of 

 substrates (Nason, 1956). Dicumarol, which inhibits menadione reductase, is a potent 

 inhibitor of E. coli nitrate reductase. 



Molybdenum probably act as an electron acceptor since the enzymatic oxida- 

 tion of FMNH2 by molydate takes place under anaerobic conditions in the ab- 

 sence of nitrate. Likewise, chemically reduced molybdate enzymatically reduces 

 nitrate to nitrite. 



The TPNH-nitrate reductase of Neurospora is a molybdenum flavoprotein (Nason and 

 Evans, 1953). A metal and a flavin also mediate the reduction of nitrite to hydroxylamine 

 and of hydroxylflTwme to ammonia by pyridine nucleotide linked reductases of Neurospora : 

 (Nason et al., 1954; Zucker and Nason, 1955). 



FAD-Metal 

 2 DPNH + 2H^ + HNO2 > NH2OH + H2O + 2DPN^ 



FMN-Metal 



(DPNH + ff + NH2OH > NH3 + H2O + DPN^ 



or TPNH) 



The studies of Sato and collaborators have implicated the cytochrome pigments in the 

 reduction of nitrate by E. coli and other bacteria (Taniguchi et al., 1956; Sato, 1956). 

 The E. coli enzyme is found in the particulate matter of the cell and may be sedimented 

 by centrifuging cell free extracts at 22,000 g for 30 min. The partially purified enzyme 

 catalyzes the anaerobic reduction of nitrate or nitrite by reduced methylene blue, formate, 

 or DPNH. Succinate, lactate, a-glycerophosphate, or formate are also effective hydrogen 

 donors. Spectroscopic observations indicate that the addition of nitrate to cell free extracts 

 (in the absence of oxygen) causes the characteristic bands of reduced cytochrome b to 

 fade. This anaerobic oxidation of cytochrome is prevented by cyanide at concentrations 

 which inhibit the reduction of nitrate by the same extracts. Likewise, 2-heptyl-4-hydroxy- 

 quinoline-N-oxide (HOQNOj, a substance which inhibits electron transport between 



Literature p. 124 



