448 IX. IIEMATIN ENZYMES, II 



Models. Resonant systems similar to porphyrins, as well as their metal 

 complexes, can act as activators of molecular hydrogen at temperatures above 

 ■-200° C. Calvin, Polanyi, and co-workers {S'.Xi) have shown that phthalo- 

 cyanine and copper phthalocyanine: (a) enal)le deuterium to interchange 

 with the hydrogen of water; {b) exchange part of their own hydrogen for 

 deuterium; and (c) catalyze the "Knallgas" reaction. Not all preparations 

 were found to be active, however {21-'j8a). 



Phthalocyanines as well as porphyrins and hematins also catalyze the 

 transformation of para- into orthohydrogen {()o7). The activation energy 

 of the disruption of the H — H bond in the presence of these catalysts is so 

 small that the process might be observable at physiological temperatures if 

 the catalysts were spread over a large surface area. 



Chemical nature of hydrogenase. These observ^ations and the inhib- 

 itor experiments indicate that the enzyme is a ferrous heme compound, 

 which functions without a change of valency and is inactive in its 

 ferric form. 



There can be no doubt that it is an iron compound. Waring and 

 Werkman (2960) have recently shown that Aerobader indologenes, 

 made deficient in iron by cultivation in a medium freed from iron by 

 treatment with 8-hydroxyquinoline, had a very low hydrogenase 

 activity. 8-Hydroxyquinoline removes copper as well as iron, but 

 on replacement of adequate amounts of the former the organism is 

 still unable to produce the enzyme. In a medium poor in iron, 

 Clostridium welchii develops a pure lactic acid fermentation and 

 becomes unable to produce acetic acid, butyric acid, and hydrogen 

 gas {2105), reactions for which hydrogenase is necessary. 



Nevertheless, its nature as a heme compound cannot yet be con- 

 sidered proved, particularly since Gaffron {97Jf.) has shown that the 

 hydrogenase of green algae is inhibited by o-phenanthroline.* While 

 this substance is an excellent complex former with iron and can 

 remove iron even from ferricyanide, it does not react with normal 

 heme compounds, in which the iron is inaccessible to the chelating 

 influence of such reagents. There remains the possibility that hydro- 

 genase is a bile pigment heme compound, since the iron of these is 

 loosely bound. 



5.2. Nitrogen Fixation 



In 1928 Meyerhof and Burk {1933) observed an inhibition of nitrogen 

 fixation by high oxygen pressure. Later, Wilson and collaborators {l7Jf6, 

 2149,309o-309S,313!)) demonstrated that root nodule bacteria as well as 

 Azotobacter, i.e., nitrogen-fixing organisms, contain hydrogenase which is 

 inhibited by oxygen. Hydrogenase activity was measured by the "Knallgas" 



* The methylene blue reduction by molecular hydrogen in the presence of hydro- 

 genase is, however, not inhibited by o-phenanthroline (Still, private communication). 



