Phosphines are ribonucleotide reductase reductants that act via C-terminal cysteines similar to thioredoxins and glutaredoxins

Abstract

Ribonucleotide reductases (RNRs) catalyze the formation of 2′-deoxyribonucleotides. Each polypeptide of the large subunit of eukaryotic RNRs contains two redox-active cysteine pairs, one in the active site and the other at the C-terminus. In each catalytic cycle, the active-site disulfide is reduced by the C-terminal cysteine pair, which in turn is reduced by thioredoxins or glutaredoxins. Dithiols such as DTT are used in RNR studies instead of the thioredoxin or glutaredoxin systems. DTT can directly reduce the disulfide in the active site and does not require the C-terminal cysteines for RNR activity. Here we demonstrate that the phosphines tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THP) are efficient non-thiol RNR reductants, but in contrast to the dithiols DTT, bis(2-mercaptoethyl)sulfone (BMS), and (S)-(1,4-dithiobutyl)-2-amine (DTBA) they act specifically via the C-terminal disulfide in a manner similar to thioredoxin and glutaredoxin. The simultaneous use of phosphines and dithiols results in ~3-fold higher activity compared to what is achieved when either type of reductant is used alone. This surprising effect can be explained by the concerted action of dithiols on the active-site cysteines and phosphines on the C-terminal cysteines. As non-thiol and non-protein reductants, phosphines can be used to differentiate between the redox-active cysteine pairs in RNRs.

A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover

“…Because two C-terminal cysteine residues are known to be the acceptors of electrons from glutaredoxins and thioredoxins in the class I and NrdJm enzymes, the role in electron transfer of the S. nassauensis NrdJd CRD and its six cysteine residues was investigated. Earlier studies with alternative reducing systems have demonstrated that the phosphine TCEP specifically reduces the C-terminal cysteine residues of class I RNR, whereas DTT can reduce the enzyme active site directly ( 19 ). In this study, S. nassauensis NrdJd-wt and NrdJdΔCRD were tested with both reductants ( Fig.…”

“…Although sequence similarity between the CRD of most NrdJs on one side and the C termini of class I RNRs and monomeric NrdJs on the other side is limited to a single pair of cysteines, the mechanism appears comparable ( 4 , 5 , 14 ). This is supported by the inability of NrdJdΔCRD to accept TCEP as terminal reductant, whereas activity is retained with DTT, the latter being small enough to directly reduce the active site and the former is not ( 19 ).…”

Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction

“…Activity of TVNrdJm was tested with the artificial reductants DTT and TCEP. While DTT is able to reduce the active site cysteine pair directly, TCEP can only work via the C-terminal cysteine pair (Domkin & Chabes, 2014; Loderer et al, 2017). The NrdJm exhibited 33% higher activity with TCEP compared to DTT at their respective maxima (Fig.…”

Peer Review #3 of "Non-host class II ribonucleotide reductase in Thermus viruses: sequence adaptation and host interaction (v0.1)"

“…Activity of TVNrdJm was tested with the artificial reductants DTT and TCEP. While DTT is able to reduce the active site cysteine pair directly, TCEP can only work via the C-terminal cysteine pair (Domkin & Chabes 2014;Loderer et al 2017). The NrdJm exhibited 33% higher activity with TCEP compared to DTT at their respective maxima (Figure 3 A).…”