CROSS-LINKING AND N-(1-PYRENYL)MALEIMIDE LABELING OF CYSTEINE MUTANTSOF PROTON-PUMPING PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase of Escherichia coli is a prot on pump composed of two subunits (alpha and beta) organized as an alph a(2) beta(2) tetramer. The enzyme contains seven cysteine residues, fi ve in the alpha-subunit and two in the beta-subunit. The reaction of t hese residues with the cross-linking agent cupric 1,10-phenanthrolinat e and with the fluorescent thiol reagent N-(1-pyrenyl)maleimide was in vestigated in mutants in which one or more of these cysteine residues had been mutated to serine or threonine residues. Mutation of alpha Cy s395 and alpha Cys397 prevented disulfide bond formation to give the c rosslinked alpha(2) dimer. We concluded that the two alpha-subunits of the holoenzyme interface in the region of these two cysteine residues . Pyrenylmaleimide reacted with detergent-washed cytoplasmic membrane vesicles containing high levels of transhydrogenase protein to show ch aracteristic fluorescence emission bands at 378-379, 397-398, and 419- 420 nm. At higher ratios of pyrenylmaleimide:transhydrogenase (>5:1) a nd longer times of reaction, an eximer band at 470 nm was formed. This was attributed to interaction between noncovalently bound molecules o f pyrenylmaleimide. The cysteine residues of the beta-subunit (beta Cy s147 and beta Cys260) were covalently modified by pyrenylmaleimide. be ta Cys147 reacted more strongly than beta Cys260 with the fluorophore, and the pyrene derivative of beta Cys147 was more accessible to quenc hing by 5-doxylstearate, suggesting a proximity to the surface of the membrane. Covalent modification of beta Cys260 resulted in inhibition of enzyme activity. The inhibition was attributed to the introduction of the bulky pyrene group into the enzyme.