Process of Metabolic Evolution 557 



some of them are devoid of glycolysis system. Chief utilizable substrates for 

 their growth are lactate, pyruvate, malate, formate, hydrogen etc. Utilization 

 of succinate is rare [5] and the presence of the tricarboxyhc acid cycle has not 

 been known. These metaboHc main features are decided by the low potential 

 nature of sulphate reduction. Desulfovibrio does not grow without sulphate even 

 in the presence of lactate or other organic substrates (except pyruvate) [6]. 

 These data place the physiological characters of sulphate reduction just between 

 respiration and fermentation. 



The mechanism of nitrate reduction by bacteria has been studied for a long 

 time. In 1937, Yamagata [7], a Japanese biologist, proposed a scheme for 

 nitrate-reducing systems, according to which the reduction took place by the 

 co-operative action of a reductase specific for nitrate, dehydrogenase systems 

 offering hydrogen from substrate and intermediary hydrogen carriers. Egami, 

 Sato, Taniguchi and their co-workers studied nitrate reduction by several types 

 of bacteria, and stated that nitrate, nitrite and hydroxylamine were reduced in 

 a halotolerant bacterium and that each step of the reduction was catalysed by 

 nitrate reductase, nitrite reductase and hydroxylamine reductase respectively. 

 They were extracted and separated from each other and their nature was studied 

 [8]. The system is indicated schematically in scheme i : 



Scheme I 



Intermediary hydro- 

 Substrate -> Dehydrogenase -> gen carriers 



(Cytochrome h) 



-Nitrate reductase ->N03 

 -Nitrite reductase ^N02 

 -Hydroxylamine reductase^ 



NHaOH 



Besides flavins or synthetic dyes which serve as the hydrogen carriers in the 

 cell-free systems, Egami & Sato found that cytochrome played a rôle as the 

 carrier. Also in the cell suspension cytochrome is reduced by addition of sub- 

 strates and oxidized anaerobically by nitrate as well as by oxygen. The results 

 of inhibition experiments also show the participation of the cytochrome as the 

 carrier in the reduction. This was the first proposal that cytochrome partici- 

 pated in anaerobic oxidoreduction [9]. 



Such behaviour of cytochrome suggests not only resemblance of the electron 

 transport system of nitrate reduction to that of respiration, but also energy 

 yielding by the oxidation with nitrate in anaerobiosis. When cells of E. coli 

 oxidized formate with oxygen in the presence of phosphate labelled with ^ap^ 

 the radioactivity was observed in the washed cells after the reaction period. 

 Similar results were obtained also in the course of anaerobic nitrate reduction 

 [10] (Table 3). 



Incorporation of ^^p indicates active transfer of phosphate and presumably 

 the occurrence of oxidative phosphorylation. So, the possible formation of 

 energy-rich phosphate bonds by electron transfer in the course of oxidation by 

 nitrate as well as by oxygen can be considered. 



Besides this type of nitrate reduction, so-called 'nitrate respiration', another 

 type of non-assimilatory nitrate reduction is found in anaerobes, e.g. Clostridium 



