Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide.
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.
D004251:DNA Transposable Elements; D005420:Flavoproteins / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000032:analysis; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013053:Spectrophotometry
D004251:DNA Transposable Elements; D005420:Flavoproteins; D009249:NADP; D010088:Oxidoreductases; C021551:mercuric reductase