Light Sensitivity of Lactococcus lactis Thioredoxin Reductase

The thioredoxin system has evolved in all kingdoms of life acting as a key antioxidant system in the defense against oxidative stress. The thioredoxin system utilizes reducing equivalents from NADPH to reduce protein disulfide targets. The reducing equivalents are shuttled via a flavin and redox active dithiol motif in thioredoxin reductase (TrxR) to reduce the small ubiquitous thioredoxin (Trx). Trx in turn regulates the protein dithiol/disulfide balance by reduction of protein disulfide targets in e.g. ribonucleotide reductase, peroxiredoxins and methionine sulfoxide reductase. The glutathione system is an alternative thiol-based antioxidant system, but the glutathione biosynthesis system is not present in all organisms.

This thesis focuses on the TrxR from the lactic acid bacteria (LAB) model organism Lactococcus lactis ssp. cremoris MG1363, a strain that is glutathione- and catalasenegative, thus expected to rely mainly on the Trx system for thiol-disulfide control. L. lactis is an important industrial microorganism used as starter culture in the dairy production of cheese, buttermilk etc. and known to be sensitive to oxidative stress. The L. lactis TrxR (LlTrxR) is a homodimeric flavoenzyme with each monomer consisting of a FAD- and a NADPH domain. In this type of low molecular weight (LMW) TrxR the NADPH domain rotates 66° relative to the FAD domain in order to complete a catalytic cycle. The TrxR thus exists in two conformations, referred to as FO- and FR-conformation. In the FRconformation NADPH reduces the FAD co-enzyme, followed by rotation to the FOconformation in which FADH2 reduces the disulfide in the redox active motif of TrxR. The human TrxR belongs to the high molecular weight (HMW) TrxR involving a selenosulfide pair and functions in a different way than the LMW TrxR, which potentially makes LMW TrxR a therapeutic target.

LlTrxR has been shown to be photo-inactivated by visible light exposure (λmax = 460 nm), which has not been reported in other TrxR and the feature was not observed using the E. coli homolog (EcTrxR) as control. The inactivation coincides with a shift in the absorbance spectrum of the tightly bound FAD co-enzyme and oxidation of the methyl group of the isoalloxazine ring, as determined by MS. The extracted FAD from photo-inactivated LlTrxR also displayed a positive result in a dinitrophenylhydrazine (DNPH) test, indicating the presence of a carbonyl group, i. e. an aldehyde. LlTrxR reduces O2 in the presence of NADPH faster than the EcTrxR and the photo-inactivation is lowered at semi-anaerobic conditions and in the presence of iodine a well-known quencher of photoexcited triplet state flavin.

The present PhD study was initiated in order to identify the underlying functional and structural mechanisms behind this light sensitivity. Crystal structures of photo-inactivated LlTrxR revealed oxidative damages over the course of light exposure. An increased electron density was observed around the carbon-7α of the isoalloxazine ring and to a minor degree around the carbon-8α. The Tyr237 in the vicinity of the flavin was shown to develop increased electron density at C3 position (ortho to the hydroxyl group) as a function of light exposure and was verified by MS to be associated with a +16 Da mass shift, consistent with formation of 3,4-dihydroxyphenylalanine (DOPA). A novel FAD si-face open space was identified in all structures of LlTrxR and predicted to accommodate O2, thus acting as an oxygen pocket. This model explains how the protein-bound FAD can function as a de facto photosensitizer, generating reactive oxygen species (ROS) upon light exposure. Reaction mechanisms accounting for the observed oxidations on FAD and Tyr237 were proposed with the photo-excited isoalloxazine ring generating a superoxide radical (O2•-) at the si-face oxygen pocket. The one-electron deficient isoalloxazine cation can then oxidize Tyr237, which upon deprotonation forms a Tyr phenoxyl radical, a target of superoxide at the C3 position, accounting for the DOPA formation. The superoxide radicals can in addition react with the deprotonated form of carbon-7α of the isoalloxazine, which via a Russel mechanism accounts for the observed aldehyde formation. Another distinct feature of LlTrxR is that it crystallizes mainly in FR-conformation, both with and without NADP+ co-crystallization. LlTrxR was only obtained in FO-conformation in reduced environment during the crystallization in the presence of DTT and absence of NADP+. Interestingly, a mixed FO-FR conformation of the homodimer was also obtained in the presence of phosphate, indicating that the two monomers might function asynchronically. The oxygen pocket is arising from the Met43 bending way from the si-face towards Pro15. Three methionines, Met18, Met43 and Met67 are bending towards the residue of Pro15 constituting (what in this work is referred to as) a methionine-proline motif.

Identification of key residue surrounding the oxygen pocket makes it possible to predict TrxR from other organisms harboring the FAD si-face oxygen pocket, including organisms such as Bacillus subtilis (BsTrxR) and pathogens such as Staphylococcus aureus (SaTrxR), Streptococcus pyogenes and Bacillus anthracis. A comparative photo-inactivation of TrxR from L. lactis, S. aureus and B. subtilis reveals that SaTrxR and BsTrxR are much less sensitive to light-inactivation than LlTrxR, though SaTrxR exhibited a similar rate of O2 reduction in the presence of NADPH as LlTrxR. Light exposure of L. lactis cell extract showed a prominent drop in TrxR activity and after 12 h about 35% of the LlTrxR remained. Preliminary experiments of light exposed living L. lactis cells kept at 4°C, indicate that light exposure is in fact lethal, under the applied conditions. Cell extracts from the same 17 h in vivo irradiated cells showed ~14% remaining TrxR activity. The present investigation shows that TrxR light sensitivity might be a widespread phenomenon among bacteria, particular within the phylum of Firmicutes. This feature can potentially be exploited in clinical light therapy, e.g. in targeted blue light therapy of selected drugresistant pathogenic bacteria.