DIHYDROXY-ACID DEHYDRATASE, A [4FE-4S] CLUSTER-CONTAINING ENZYME IN ESCHERICHIA-COLI - EFFECTS OF INTRACELLULAR SUPEROXIDE-DISMUTASE ON ITSINACTIVATION BY OXIDANT STRESS

Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is report ed to be facilely inactivated by oxidant stress, To directly assess th e biological effects in vivo of superoxide dismutase (SOD) on the oxid ant sensitivity of DHAD, we used an Escherichia coil K-12 parent strai n (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD, In the K-12 parent strain half the cellula r DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 mu M aerobic nitrofurantoin, or about 5 mu M aerobic paraquat (PQ) and in about 1 min at 10 mu M aerobic PQ, Oxygen and metabolism were require d for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-fr ee extracts did not restore DHAD activity, The K-m was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respective ly, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponentia l, aerobic growth, Thus, active site oxidative impairment of individua l enzyme molecules apparently was all-or-none, DHAD activity was great ly decreased when measured in extracts made from strains that lacked b oth SODs unless SOD was added to cell suspensions before extracts were made, DHAD was more sensitive in strains lacking both SODs than in th e parent strain to inactivation by aerobic PQ and HBO, Anaerobic (comp ared to aerobic) growth increased DHAD specific activity by 20% or les s in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHA D was increased 60%, DHAD was partially inactivated by the oxidant str ess of aerobic growth, but remained in a form detectable by DHAD antib ody, and the ratio of active to inactive DHAD decreased greatly in cel ls lacking SOD, Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low level s of PQ. (C) 1995 Academic Press, Inc.