Proton-coupled Electron Transfer Pathway In Human Ribonucleotide Reductase

Ribonucleotide reductase (RR) catalyzes the rate-limiting step in DNA precursor synthesis:the reduction of ribonucleotides to deoxyribonucleotides in all living cells. The key role of RR in DNA synthesis, cell proliferation, and malignant transformation makes it an important target for anticancer drugs. Studies on structure and function of RR will help clarify the mechanism of RR regulation and design novel RR inhibitors for cancer treatment. RR is divided into three classes based on different metal cofactors for catalytic activity. Class Ⅰ RR is further divided into three subclasses (Ia, Ib, and Ic) according to their polypeptide sequence homology and overall allosteric regulation behavior. Human, mouse, and E.coli RRs belong to Class Ia. Most of the studies on structure and function of the class Ia RRs have been performed on E. coli RR, which is a tetramer composed of two homodimeric subunits designated R1(large subunit) and R2(small subunit). In E.coli RR, reversible proton-coupled electron transfers (PCET) for radical transport across a distance about35A between R2(·Tyr122→Trp48→Tyr356) and R1(Tyr731→Tyr730→·Cys439) are critical for the enzyme catalysis. In R2, proton acceptors positioned close to these redox active a分钟o acid residues couple distant electron transfer reactions to short-range proton transfers. Tyr356in R2locates in the interface of Rl and R2, and the mechanism of the PCET between Trp48, Tyr356, and R1has long been a mystery. In this study, using the small subunit M2of human RR (hRRM2) as a model system, we seek to identify essential residues for the PCET pathway between Trp102(the counterpart of Trp48in E.coli) and Tyr369(the counterpart of Tyr356in E.coli). Expression plasmids of native, truncate, mutated hRRM2were constructed, the proteins were expressed in E.coli and highly purificated for X-ray diffraction crystal structural and functional analysis. We mutated6surface residues within5A around Trp102to alanine and found that only E106A was completely inactive. A more comprehensive site-directed mutagenesis found that E106D possessed about10%of the enzyme activity of native RR protein, while all other mutants of Elu106, including E106Q, E106A, E106R, E106K, E106L, and E106Y, were completely inactive, indicating Elu106was essential for RR activity.Surface plasmon resonance (SPR) measurements demonstrated that E106D and E106Q mutations did not affect hRRM1-hRRM2interaction. The X-ray diffraction crystal structures of the mutants E106A, E106D, and E106Q showed very little structural perturbation compared to the native protein, indicating that Elu106plays a catalytic rather than structural role. Electron paramagnetic resonance (EPR) measurements showed that the formation of the stable tyrosyl radical in E106A.E106Q and E106D mutants were similar with the native protein, which demonstrates that Elu106was not involved in the tyrosyl radical formation. Furthermore, the enzyme suicide2-azido-2-deoxy-CDP (CzDP) assays confirmed that the radical transport is obliterated by the mutations of E106A and E106Q, but E106D was still partially capable of mediating the PCET. Based on the crystal structures of hRRM1and hRRM2, the holoenzyme structure model of hRR was builded, and the enzyme dynamic anslysis and theoretical calculations were further conducted by computer-asistant methord. All these data demonstrated that Elu106is a key residue for hRR enzymatic activity, probably functions as a proton mediator for Tyr369in the PCET pathway of hRRM2. Together, we propose that Elu106participats in PCET pathway as a a proton mediator for Tyr369at the C-terminus of hRRM2as well as the interface of the small and large subunits of hRR.Meanwhile, for the first time, we present a holoenzyme structure model of hRR base on the crystal structures of hRRM2and hRRMl. All these findings provide a new and valuble insight into the molecular mechanisms of PCET in hRR, and help design novel hRR inhibitors as anticancer drugs.