446 IX. HEMATIN ENZYMES, II 



5. ENZYMES POSSIBLY OF HEMOPROTEIN NATURE 



5.1. Hydrogenase 



A very interesting enzyme, hydrogenase, which catalyzes reactions with 

 molecular hydrogen has been found in a variety of microorganisms, E. coli, 

 Proteus, Acetobader peroxydans, Azotobacter, lactic acid bacteria, root nodule 

 bacteria {1665), and organisms isolated from river mud {cf. 2625). It also 

 occurs in butyric acid fermenters and Clostridium irelchii and participates in 

 the photosynthetic processes. 



The primary reaction which it catalyzes is: H2 ;=^ 2 H+ + 2 e {10Jf5). A 

 bright metal electrode in contact with a suspension of E. coli and hydrogen 

 acts as a hydrogen electrode; Green and Stickland (1045) have measured the 

 oxidation-reduction potential of the enzyme of E. coli in the presence of 

 hydrogen with methylviolinogen as oxidation-reduction indicator and have 

 found Eo' — — 0.40 at pH 7, 30° C. Molecular hydrogen activated by this 

 enzyme is able to reduce a great variety of hydrogen acceptors, e.g., molecular 

 oxygen, catalyzing the "Knallgas" reaction: 



2 H2 -F O2 -» 2 H2O 



(2626,3073) ; carbon dioxide to formic acid as well as to methane (2626,2629, 

 3119); acetic acid (3079); nitrate to nitrite (2626); sulfate to hydrogen 

 sulfide; phosphate; and organic hydrogen acceptors such as fumarate and 

 methylene blue (10^5), although the latter perhaps not directly (1297). For 

 a review of the older literature the reader is referred to the papers of Stephen- 

 son and Stickland, and particularly to Stephenson's book (2625). 



Farkas and collaborators (738) and Hoberman and Rittenberg (1297) have 

 studied the hydrogenase-catalyzed reaction: H. -f- D2O ;=^ HD -|- DOH 

 (D = deuterium). It is of interest to note that colloidal platinum is perhaps 

 even a better model for the reactions catalyzed by hydrogenase than for the 

 catalatic decomposition of hydrogen peroxide; thus it catalyzes the equi- 

 librium: CO, + H. ;=i HCO2H (Bredig and Carter, 33^), as well as the 

 "Knallgas" reaction. 



It is to be expected that the enzyme also catalyzes the inverse reactions, 

 e.g., the spHtting of formic acid; HCO2H -+ CO2 4- H2. Stephenson and 

 Stickland (2629,2663) have assumed that this reaction is caused by a distinct 

 enzyme, hydrogenlyase, but it is now probable that this is identical with 

 hydrogenase (898,1297,2080). It is also very likely that the development of 

 molecular hydrogen in fermentations, e.g., butyric acid fermentation, is 

 catalyzed by hydrogenase (1507,1513,1590,2105). ^ 



Enzyme preparations. Crude cell-free extracts of hydrogenase have been 

 obtained by Wilson and collaborators (1665) from Azotobacter vinelandii by 

 grinding with powdered glass in M/\o phosphate buffer of pH 7, and from 

 E. coli by Kalnitsky and Werkman (14-60,^61) and by Still and co-workers 

 (115). By freezing and drying in vacuo the enzyme was obtained in a stable 

 form. Inhibition experiments, supported by studies on the iron metabolism 

 of microorganisms and on certain models, suggest that the enzyme may be 

 a hematin compound or a closely related substance. 



