560 M. ISHIMOTO AND F. EGAMI 



yielding systems, different from substrate level phosphorylation. The ability of 

 phosphor>'lation in the electron transfer should provide the energetical base of 

 autotrophic life. In the primaeval reaction cytochrome participated in the 

 reaction with hydrogen-hydrogenase and with reductase for sulphate, nitrate 

 or other inorganic compounds. 



In the second step cytochrome became connected with photo-oxidation, 

 which induced high redox potential and prepared conditions for oxidation of 

 various types of compounds. 



In the third step, respiration started and developed with the appearance of 

 oxygen in the atmosphere. The complexity of the respiratory system corresponds 

 to its higher efficiency in energy yielding. 



Enzyme systems of nitrate and sulphate reduction for assimilation differ 

 greatly from those for energy yielding. Assimilatory reduction which is observed 

 in higher plants, Neurospora and bacteria in some conditions, is carried out by 

 molybdenum-containing fiavoproteins. The reduction in seedlings of bean has 

 already been mentioned. 



Sulphate reduction for assimilation leading to organic sulphur compounds 

 takes place widely in micro-organisms and plants, but only few data on the 

 mechanism were offered. Recently Lipmann discovered the activation of sul- 

 phate by adenosine triphosphate in extracts of Neurospora [25], which might 

 be a preparatory step for sulphate reduction. The path of sulphite reduction 

 to cysteine in mammalian tissues is not through direct sulphite reduction to 

 inorganic sulphide but through reduction of cysteine sulfinic acid, which is 

 formed from pyruvate and sulphite through addition and transamination [26]. 

 Intermediary formation of inorganic sulphide in the process of sulphate assimi- 

 lation was denied by several authors in the field of biochemical genetics [27]. 



When and how did these differences arise in the path and enzyme systems 

 between assimilatory and non-assimilatory reduction of nitrate and sulphate ? 

 Before the appearance of oxygen on the Earth, the most abundant inorganic 

 forms of nitrogen and sulphur were possibly ammonia and sulphide. Accordingly, 

 hving organisms could utilize ammonia and sulphide for body building, and 

 necessity of reduction for assimilation might arise only after the appearance of 

 oxygen in the atmosphere and consequent decrease of ammonia disappearance 

 and of sulphide by oxidation. 



The first type of nitrate reduction might be one, like nitrate fermentation 

 in Clostridium or cotyledons, and has effects on the oxidation of substrate and 

 on the elevation of potential in the organisms. Along with the evolution of 

 cytochrome, there appeared the coupling of nitrate reductase with the cyto- 

 chrome system. Assimilatory reduction evolved from the first type of nitrate 

 reduction independently from the systems of nitrate respiration. 



Studies on enzymic mechanisms of nitrogen fixation have not given conclu- 

 sive results and little information on its evolution has been obtained. But the 

 facts, that biogenic nitrogen gas was not present in the atmosphere and that 

 many anaerobes as well as aerobes perform nitrogen fixation, may indicate the 

 precedence of that process before respiration. Denitrification, which is carried 

 out by facultative anaerobes, can be considered to appear later. 



