PROBING THE ROLE OF ELECTROSTATIC FORCES IN THE INTERACTION OF CLOSTRIDIUM-PASTEURIANUM FERREDOXIN WITH ITS REDOX PARTNERS

The ability of several low-potential redox proteins to mediate electro n transfer between Clostridium pasteurianum pyruvate-ferredoxin oxidor eductase and hydrogenase has been evaluated in a coupled enzymatic ass ay. The active electron mediators, whatever their structure, must have a reduction potential compatible with the two enzymes, but for protei ns of similar potentials, a marked specificity is displayed by 2[4Fe-4 S] ferredoxins of the clostridial type. Such ferredoxins are small pro teins exchanging electrons with many enzymes involved in the metabolis m of anaerobic bacteria. The forces underlying the interactions of fer redoxin with hydrogenase and pyruvate-ferredoxin oxidoreductase have b een examined with an emphasis on electrostatics: site-directed mutagen esis experiments have been used to individually convert all conserved glutamates and aspartates of C. pasteurianum ferredoxin into either ne utral or positively charged amino acids. Also, up to four of these res idues have been replaced simultaneously. The biological activities of the resulting variants depend very little on the number and the distri bution of the anionic side chains on the surface of the ferredoxin. On ly those molecular forms for which the immediate environment of the cl usters is perturbed, independently of the charge distribution, display variations in their catalytic properties. It is concluded that electr on transfer between C. pasteurianum 2[4Fe-4S] ferredoxin and its partn ers is far less dependent on electrostatic interactions than in many o ther well-documented electron transfer systems.