Surface enhanced vibrational spectroscopic evidence for an alternative DNA-independent redox activation of endonuclease III

Abstract

Surface enhanced vibrational spectro-electrochemistry of endonuclease III provides direct evidence that the [4Fe-4S] cluster is responsible for the enzyme redox activity, and that this process is not exclusively DNA-mediated, as currently proposed. We report the first surface enhanced resonance Raman spectrum of a [4Fe-4S] 2+ cluster containing enzyme.Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium, which can withstand 200-fold higher irradiation doses than other bacteria. 1,2 The resistance mechanism is not known, but an efficient DNA repair machinery is considered to play a key role in it. Endonuclease III (EndoIII) is a [4Fe-4S] cluster containing DNA repair enzyme from the helix-hairpin-helix family of DNA glycosylases, crucial for removal of oxidation damaged bases in DNA. 3,4 Early evidence indicated that Fe-S clusters in these enzymes are not amenable to oxidation or reduction in solution and suggested structural or regulatory roles of the cofactor. 5,6 More recent electrochemical studies of glycosylases immobilised on DNA-modified electrodes indicated that the [4Fe-4S] cluster is activated towards oxidation upon binding to DNA. It was proposed that the cluster exerts a function in DNAmediated signalling for detection of DNA lesions, 7-12 but based on data obtained by electrochemical approaches, which cannot provide any information about the molecular origin of the electrochemical signal. Here we employed surface enhanced resonance Raman (SERR) spectroscopy and surfaced enhanced IR absorption (SEIRA) spectro-electrochemistry to demonstrate that the cluster of the immobilised EndoIII from D. radiodurans (DrEndoIII) is prone to reduction, which is not necessarily DNA-mediated.DrEndoIII was electronically coupled to metal electrodes modified with bifunctional alkanethiol-based self assembled monolayers (SAMs). 13 The cluster integrity of DrEndoIII immobilised on the 11-mercapto-undecanoic acid (MUA)-coated nanostructured Ag working electrode was probed by SERR spectroscopy. For DrEndoIII, which has a broad electronic absorption band centered at 410 nm, both the plasmonic and the resonance enhancement conditions are well matched using 413 nm excitation. 14 The negatively charged MUA surface readily interacts with the highly positively charged DNA binding site of DrEndoIII (vide infra) that includes the active site pocket and the [4Fe-4S] cluster (Fig. S1, ESI †), which is in glycosylases optimised for a fast and precise accommodation of the negatively charged DNA substrate. 4,[15][16][17] Upon protein immobilisation and removal of the loosely bound molecules by rinsing with buffer, the functionalised flat disk Ag electrode was inserted into a pre-cooled cryostat that was rapidly cooled to 77 K. The SERR spectrum of the Ag-MUADrEndoIII construct was measured at 77 K since the intrinsically weak RR bands of Fe-S clusters can only be detected at low temperatures. 18,19 The spectrum shows well defined modes at 337, 363 and 384 cm À1 (Fig. 1a), indicative of a [4Fe-4S] clus...

Resonance Raman spectroscopy of Fe–S proteins and their redox properties

“…Spectra were measured with 413 nm excitation at 77 K using 1.5 and 8 mW in the SERR and RR experiments, respectively.Reproduced from Ref. [11] with permission from The Royal Society of Chemistry…”

“…The large body of work on synthetic clusters and simple monocluster containing proteins, mainly those involved in electron transfer (ET) (vide infra 3.1) led to a well-established assignment of their vibrational spectra and paved the way to studies of more complex systems [ 3 – 10 ]. More recently, RR has been shown to be capable of providing fine details on transient processes that involve Fe–S proteins [ 3 – 10 ], and has been extended to surface enhanced RR (SERR) to characterize immobilized Fe–S enzymes interacting with physiological substrates [ 11 , 12 ]. Here, we aim to present an overview of RR studies on structurally and functionally different Fe–S proteins, highlighting the type of information that can be extracted from RR spectra and in parallel, discuss their thermodynamic properties (e.g., reduction potential).…”

“…After “Introduction”, we will first briefly describe the basics of RR spectroscopy of Fe–S proteins, together with the most commonly used approaches for determination of their redox properties. Then we will focus on the major contributions of RR spectroscopy in the studies of diverse Fe–S proteins, such as those that participate in ET [ 1 , 13 – 21 ], DNA repair [ 11 ], biogenesis of Fe–S clusters [ 3 , 22 – 30 ] and heme cofactors [ 31 ], substrate binding and activation, S-donation and catalysis [ 32 – 35 ], and regulation of gene expression [ 5 , 8 , 10 , 36 – 39 ]. We will conclude with several examples of recent RR studies of enzymes carrying complex polychromophoric clusters [ 7 , 40 , 41 ].…”

Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA

“…It was recently reported that carboxylic acid monolayers had a similar activating effect as DNA, although, in contrast with the above model, they identified the relevant couple as the [4Fe4S] 2+/+ rather than the [4Fe4S] 3+/2+ couple (16). With respect to the latter point, the high potential of the reversible DNA-bound signal on both gold and HOPG, EPR spectroscopy of oxidized DNA bound EndoIII and MutY, and the observation of both couples in the expected potential regimes on HOPG support the original [4Fe4S] 3+/2+ assignment (6,8,15).…”

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans

“…Initially it was suggested to possess only a structural role (Fu et al, 1992) and later that it is involved in redox regulated DNA mediated signaling (Boal et al, 2005). Recently the cluster was also proposed to act as a redox centre that could be activated by charged entities other than DNA, suggesting a potential role in processes related to the organism's defense against reactive oxygen species (Moe et al, 2015). There is no evidence so far that the cluster may be directly involved in the catalytic process.…”