The physical and catalytic properties of hydrogenase II of Clostridium pasteurianum. A comparison with hydrogenase I.

Abstract

Hydrogenase I1 of Clostridium pasteurianum is a monomeric protein of Mr = 53,000 containing 8 iron and 8 acid-labile sulfide atoms/mol.It is distinct from hydrogenase I from the same organism (Mr = 60,000 12 Fe and 12 Sz-/mol).Metal analyses showed that neither hydrogenase contains nickel or any other metals in significant amounts.The iron atoms of hydrogenase I1 resisted chelation by 2,Z"bipyridyl but all were susceptible when the enzyme was treated with ferricyanide.Core extrusion indicated the presence of two [4Fe-4S] clusters in hydrogenase I1 and EPR spectroscopy showed two distinct paramagnetic species which could be interpreted as one [4Fe-4S]2+(z+3'+) and one [4Fe-4SI2+('+r3+) per molecule.The absorption coefficient of Hz-reduced hydrogenase I1 at 420 nm was 23,000 M" cm" with a A4z0/A276 ratio of 0.27.There were large differences between hydrogenase I and hydrogenase I1 in the absorption spectra of the air-oxidized, H2-reduced, and dithionite-reduced forms of the enzymes.Hydrogenase I1 catalyzed Hz evolution with methyl viologen or ferredoxin as the electron carrier, and Hz oxidation with methylene blue or methyl viologen as the electron acceptor.Apparent K, values were determined for all these reactions with both hydrogenases.Hydrogenase I1 is a relatively inactive enzyme, except in the reduction of methylene blue by Hz.The pH dependencies of Hz oxidation were similar for both hydrogenases but were very different in Hz evolution.The activation energy values were much higher for Hz catalysis by hydrogenase I1 than for hydrogenase I.The two hydrogenases have the same sensitivity to inactivation by Oz but differ in their sensitivity to metal-chelating reagents and to CO. Hydrogenase I is more readily inhibited by CO but hydrogenase I1 binds CO irreversibly.From the above data, a mechanism is proposed to account for the observed differences in the catalytic activities of hydrogenase I and hydrogenase 11.The anaerobic Nz-fixing bacterium Clostridium pasteurianum produces large amounts of Hz as an end product of metabolism.These saccharolytic organisms metabolize carbohydrates to organic acids, Hz, and COz and because they lack an electron transport chain, they obtain ATP by substrate level phosphorylation.The enzyme hydrogenase (EC class 1.12) which catalyzes the reversible activation of Hz has been purified from this organism and has been characterized * The costs of publication of this article were defrayed in part by the payment of page charges.