600 YOSHIHARU ODA 



TRANSITIONARY STAGE 

 FROM ANAEROBIOSIS TO AEROBIOSIS 



It is first emphasized that the activity of the hydrogenase system shows 

 definite sensitivity and instability to Oj [15, 1 6]. In a species of lactic acid bacteria, 

 Lactobacillus casei, which is a t^'pical anaerobic fermenter, the metabolic system 

 is not influenced by the presence of Oj. Inversely, the metabolic systems of 

 Clostridium and the coli-aerogenes group containing the hydrogenase system, are 

 inactivated or fail to be formed, in the presence of O2. In short, the fact that the 

 hydrogenase system is very unstable and easily inactivated by Oo shows that it 

 has some reactivity or affinity for Oo. According to our experiments, the hydro- 

 genase system in cell-free preparation from E. coli and Azotobacter chroococcum 

 that is inactivated by Oo is autocatalytically reactivated when left in a hydrogen 

 atmosphere, but not in a nitrogen atmosphere. When at first inactivated by 

 shaking with O2 beyond the time of threshold, it is not reactivated again even in 

 a hydrogen atmosphere. It may be inferred that the inactivation of the hydro- 

 genase system by O? is not due to oxidation but to combination with O2, that is, 

 to oxygenation like that of haemoglobin [15]. 



The oxidation of H2 with ferricyanide or methylene blue as the hydrogen 

 acceptor by the hydrogenase system in cell-free preparations was inhibited by 

 CO, but the inhibition cannot be reversed by Ught. HCN partially inhibits the 

 oxidation of H2 with the dye only after preincubation with O2 but not in a 

 hydrogen atmosphere. 



Accordingly, it may be conceivable that in this system the typical iron por- 

 phyrin component is not involved, but a heavy-metal component is a cofactor. 

 As mentioned above, a flavin component might be involved in this system as 

 evidenced by the spectrum studies, and so the system may be probably a 

 flavoprotein containing heavy metal as a cofactor. 



Next, it is very important that even in the facultative anaerobes such as E. coli 

 the synthesis of the hydrogenase system occurs only upon cultivation in anaerobic 

 conditions. In this connection it should be noted that the system in the strict 

 anaerobes, such as Clostridium, is extracted almost completely in the soluble 

 cell-free system, but in the facultative anaerobes, E. coli, it is partially bound to 

 the insoluble particles and partially in the soluble state. 



After anaerobic dialysis against water this concentrated soluble cell-free 

 system from E. coli is capable of decomposing formate only after a prolonged 

 induction period. This induction phase is markedly shortened or abolished 

 when the dialysed extract is preincubated with boiled preparations from E. coli. 

 Cl. butyricum, yeast or pigeon liver. Among the variety of purified potential co- 

 factor tested, only pyruvate, diacetyl and acetyl phosphate were found to spark 

 decomposition of formate by dialysed extracts. Furthermore, under proper 

 conditions small quantities of pyruvate, diacet}'l or acet}'l phosphate are also 

 capable of sparking decomposition of formate by the 'dilute' (inactive) E. coli 

 extract but only in the presence of C/. butyricum extract. The Clostridium extract, 

 which docs not metabolize formate, appears to be required for converting the 

 added cofactor to the actual sparking intermediate. All of the above observations 



