Abstract
The flavoprotein mercuric reductase catalyzes the two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. It has now been purified from Pseudomonas aeruginosa PAO9501 carrying the plasmid pVS1. In this plasmid system, where the mer operon is on the transposon Tn501, mercuric reductase comprises up to 6% of the soluble cellular protein upon induction with mercurials. The purification is a rapid (two-step), high yield (80%) procedure. Anaerobic titrations of mercuric reductase with dithionite revealed the formation of a charge transfer complex with an absorbance maximum around 540 nm. Striking spectroscopic similarities to lipoamide dehydrogenase and glutathione reductase were observed. These two enzymes, which catalyze the transfer of electrons between pyridine nucleotides and disulfides, are flavoproteins which contain an oxidation-reduction-active cysteine residue at the active site. The expectation that mercuric reductase contains a similar electron acceptor was confirmed when it was shown that mercuric reductase has the capacity to accept four electrons per FAD-containing subunit, and that two thiols become kinetically titrable by 5,5'-dithiobis-(2-nitrobenzoate) upon reduction with NADPH. These are characteristic features of the disulfide reductase class of flavoproteins. Further similarities with at least one of these enzymes, lipoamide dehydrogenase, include the E/EH2 midpoint potential (-269 mV), fluorescence properties, and extinction coefficients of E and EH2. Preliminary observations relevant to an understanding of the mechanism of mercuric reductase are discussed.
Highlights
The expectation mechanistic similarity to otherclasses of flavoproteins. It was thatmercuricreductasecontainsasimilarelectron with greatinterest,that we observed the high acceptor was confirmed when it was shown that mer- degree of similarity to lipoamide dehydrogenase and glutathicuric reductase has the capacity to accept four elec- one reductase that we report in this paper
Air wasbubbled through the reaction mixture and gratuitous inducer for this system [9],up to 6%of the soluble through aqua regia to reoxidize and trap the ’03Hg”.Aliquots of the protein of P. aeruginosa PA09501 is mercuric reducreaction mixture were drawn with a syringe and the residual radioactivity was counted in Amersham ACS scintillation fluid
A similar NADP assay was performed on heat-treated crude extract, with the concentration of mercuric reductase estimated by enzyme activity
Summary
PURIFICATION AND CHARACTERIZATION OF A TRANSPOSON-ENCODED FLAVOPROTEIN CONTAINING AN OXIDATION-REDUCTION-ACTIVE DISULFIDE*. Anaerobic titrations of mercuric reduwcittahse dithionite revealed the formationof a charge transfer This paperdescribes arapid purification and the characterization of a mercuric reductase from Pseudomonas aeruginosa PA09501 carrying the plasmid pVS1 [9]. It was thatmercuricreductasecontainsasimilarelectron with greatinterest,that we observed the high acceptor was confirmed when it was shown that mer- degree of similarity to lipoamide dehydrogenase and glutathicuric reductase has the capacity to accept four elec- one reductase that we report in this paper These flavoentrons per FAD-containing subunit, and thattwo thiols zymes catalyze a very different reaction, the transferof elecbecomekineticallytitrableby 5,5’-dithiobis-(2-nitro- trons between pyridine nucleotides and disulfides (1 1)T.his benzoate) upon reduction wNitAhDPH. Fined as the amount ofenzyme that catalyzes the Hg’+-dependent oxidation of 1.0 pmol of NADPH per min [8]
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