title: 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.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Domkin
authors.affiliation: 1] Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden [2] Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, SE 90187 Umeå, Sweden.
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
mesh_terms: D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation; D054477:Glutaredoxins / Q000737:chemistry*; D051379:Mice; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry*; D011485:Protein Binding; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry*
chemical_list: D054477:Glutaredoxins; D010720:Phosphines; C000603174:tris(3-hydroxypropyl)phosphine; D013879:Thioredoxins; D012264:Ribonucleotide Reductases; D003545:Cysteine