Lactococcus lactis Thioredoxin Reductase Is Sensitive to Light Inactivation

Abstract

Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low-molecular weight thioredoxin reductases and display similar k cat values (∼25 s–1) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation under standardized conditions (λmax = 460 nm and 4 °C) was reduced at lowered oxygen concentrations and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the flavin adenine dinucleotide (FAD) that remained firmly bound. High-resolution mass spectrometric analysis of heat-extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to that of unmodified FAD, corresponding to the addition of one oxygen atom and the loss of two hydrogen atoms. Tandem mass spectrometry confined the increase in mass of the isoalloxazine ring, and the extracted modified cofactor reacted with dinitrophenyl hydrazine, indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen reduction are discussed in relation to other flavin modifications and the possible occurrence of enzymes with similar properties.

The structure of Lactococcus lactis thioredoxin reductase reveals molecular features of photo-oxidative damage

“…3a–f), even though the FAD co-enzyme in general has well-defined electron densities with B-factors typically in the range from 20–24 Å 2 . This observation is in accordance with our previous mass spectrometry data demonstrating formation of an aldehyde group in FAD extracted from light-inactivated LlTrxR, which was concluded to be confined to the C7α methyl group based on the spectrophotometric features16. Moreover, an increase in electron density at the meta-position (ortho to the hydroxyl group) of Tyr237, located 4.4 Å from C7α is observed as a function of light-exposure (Fig.…”

“…Besides its proposed role in photo-sensitivity the oxygen pocket might also have implications for the ability of these enzymes to reduce O 2. We have reported that LlTrxR displays a ~10-fold increased rate of O 2 reduction in the presence of NADPH as compared to EcTrxR16. LlTrxR thus shows a similar tendency to reduce O 2 as the TrxR homologue AhpF, a dedicated reductase of the peroxide scavenger AhpC in eubacteria2829.…”

“…The observed oxygen- and triplet isoalloxazine-dependent photo-inactivation of LlTrxR16 suggests that molecular oxygen is positioned in the vicinity of the FAD and interacts with the photo-excited co-enzyme. A closer look at the isoalloxazine ring indeed reveals a pocket that potentially may accommodate molecular oxygen due to its hydrophobic character.…”

“…We have previously shown that recombinant TrxR from L. lactis is inactivated by visible light in vitro , while the corresponding enzyme from E. coli is more resistant to photo-inactivation under these conditions16. In order to evaluate the sensitivity of native TrxR in an environment resembling the conditions in vivo , cell extracts of mid-late exponential phase cultures of L. lactis and E. coli K-12 were subjected to irradiation over a period of 12 h and TrxR activity was measured at different time points by use of a coupled assay with Trx, applying DTNB as the final electron acceptor.…”

“…This may explain why knock-out mutants lacking either TrxR or Trx are viable even under oxidative stress conditions15. While investigating the biochemical properties of L. lactis TrxR (LlTrxR) we discovered that the enzyme is susceptible to photo-inactivation by visible light in an oxygen-dependent manner16. This inactivation coincided with a shift in the absorbance spectrum of the tightly bound FAD co-enzyme and oxidation of an isoalloxazine methyl group.…”