Class Ic Ribonucleotide Reductases: Expansion of the Subclass and Insight into the Multi-Step Inter-Subunit Radical Translocation Mechanism
Open Access
- Author:
- Livada, Jovan
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 14, 2019
- Committee Members:
- Joseph M Bollinger, Jr., Dissertation Advisor/Co-Advisor
Joseph M Bollinger, Jr., Committee Chair/Co-Chair
Squire J Booker, Committee Member
Alexey Silakov, Committee Member
Ming Tien, Outside Member
Philip C Bevilacqua, Program Head/Chair - Keywords:
- EPR
ribonucleotide reductase
DFT
Bioinorganic - Abstract:
- Ribonucleotide reductases (RNRs) are the only known catalysts that are capable of the life essential conversion of ribonucleotides to deoxyribonucleotides. All RNRs use a transient cysteine radical (C•) to initiate the catalytic cycle. Based on the method of generation of the C•, RNRs are divided into three (I, II, III) major classes. Class III RNRs use an Fe4S4 cluster in tandem with an S-adenosyl-L-methionine radical activase to install a glycyl radical that is stable in anoxic conditions. In class III RNRs this glycyl radical is the active oxidant that forms the C•. The active oxidant in class II RNRs is 5´-deoxyadenosylcob(III)alamin which generates the C• via the reversable Co-C bond homolytic cleavage. Class I RNRs are the sole focus of this study and they contain two subunits, a smaller β subunit that contains the active oxidant and usually hosts a dimetal center, and the larger α subunit that harbors the substrate binding site and contains the key cysteine residue that is transiently oxidized during catalysis. All class I RNRs use the active oxidant in their smaller subunit β to transiently oxidize the C• in the larger subunit α that is ~35 Å away. This is a multi-step, long range electron transfer process coupled by a few local proton transfers and mediated by, at a minimum, one tyrosine residue in β and two tyrosine residues in α. Class I RNRs are further subdivided into five subclasses (a-e) that differ in four relevant properties. One, the nature of the active oxidant in β (Y•, MnIV or DOPA•), two, the presence of a dimetal cofactor (Fe/Fe, Mn/Mn, Mn/Fe, or none), three, the oxygen species required for the formation of the active oxidant (O2 or O2-•), and four, the requirement of an activase protein, NrdI. Archetypical class Ia RNRs generate the active oxidant, Y•, via the use of a β-FeII/FeII cofactor and O2. On the other hand, class Ic RNRs, the main topic of this study, use a heterobinuclear β-MnII/FeII cofactor that is activated in the reaction with dioxygen and via the β-MnIV/FeIV intermediate form the active cofactor β-MnIV/FeIII, where MnIV represents the active oxidant. RNR subclasses Ia, Ib, Id and Ie all have a minimum of three members verified by in vitro characterization, while the RNR from Chlamydia trachomatis (Ct) remains the sole verified example of class Ic ribonucleotide reductases, discovered in 2007. In this work, twelve years later, we have introduced a second class Ic RNR that uses a β-MnIV/FeIII active cofactor from Gimesia maris (Gm), a heterotroph found in various aquatic locations around the US. Rapid kinetic methods, stopped-flow and freeze quench, Mössbauer, and electron paramagnetic resonance (EPR) spectroscopies as well as activity assay analysis were all used to characterize Gm RNR as a bona fide class Ic RNR. Like Ct, Gm RNR was shown to possess a branched electron relay pathway in β that consist of two tyrosine residues and one tryptophan. Interestingly, by removing a subclass specific tyrosine residue involved in the cofactor reaction with dioxygen we were able to noticeably increase the rate of catalytic activity. This information sheds light onto the activating and catalytic electron relays in class Ic RNRs. Sequence similarity network analysis of genes that encode both for RNR α and β subunits indicate clusters of closely coevolved RNRs that belong to both class Ia and class Ic RNR subclasses. In vitro characterization of a tentative class Ia RNR from Methylococcus capsulatus (Mc) confirms that this RNR uses a Fe/Fe cluster to generate the nearby active Y• oxidant. A closely co evolved RNR whose gene sequence suggests that it belongs to the class Ic RNR subclass from Salinisphaera hydrothermalis (Sh) was also characterized and confirmed that it forms a MnIV/FeIII cofactor and that it uses MnIV as the active oxidant. Activity assays have shown that both heterologous pairs of α and β subunits of Mc and Sh RNR are able to perform bidirectional radical translocation. This represents initial evidence that the multi-step, one electron, multiple proton transfer mechanism between α and β RNR subunits is conserved between the RNR subclasses Ia and Ic. We hypothesize that this mechanism might be conserved in all class I RNRs. Additionally, we concluded that there were no adaptations required, for performing ribonucleotide reduction, made to either subunit to accommodate a complementary subunit from a different subclass except for the evolution of the β subunit to harbor a distinct active oxidant. The aforementioned multi-step, one electron, multiple proton transfer process is believed to involve a 35 Å distance between the active oxidant in β and the catalysis initiating cysteine residue in α. The active cofactor from the class Ic RNR from Chlamydia trachomatis β-MnIV/FeIII is reduced to an EPR accessible (S = 1/2) MnIII/FeIII state in the β subunit and generates a cysteinyl radical in the α active site. The radical in the larger subunit can be trapped via a known mechanism based inactivator, 2′-azido-2′-deoxyuridine-5′-diphosphate (N3UDP). The resulting reaction accumulates a stable, nitrogen-centered radical (N•) in the active site of α. Using double electron−electron resonance experiments, we have elucidated the distance between the β-MnIII/FeIII cofactor in β and N• in α to be 43 ± 1 Å. This experimental investigation not only provides the first structural information concerning the class Ic RNR holoenzyme complex but also the first direct experimental measurement of the inter-subunit RT distance in any class I RNR. In class Ia RNRs, the first step of the bidirectional, multi-step, one electron, multiple proton transfer process has been characterized as a proton-coupled-electron-transfer (PCET) reaction where the active oxidant (Y•) is reduced by a nearby organic residue in β and a local proton transfer occurs from the water ligand bound to the β-FeIII/FeIII cofactor onto the radical harboring tyrosine. The presumptive proton transfer step in class Ic, Ct RNR, in which reduction of a β-MnIV/FeIII oxidant to an EPR-active (S = 1/2) β-MnIII/FeIII state drives Cys oxidation in α, is not understood. We have used advanced electron paramagnetic resonance spectroscopies as well as density-functional theory (DFT) calculations to analyze the β-MnIII/FeIII RT product to determine the number and location of its protons. The correlation of our findings in addition to the previous structural characterization of the β-MnIV/FeIII RT-reactant state led us to identify that the acceptor for the initial proton transfer step in class Ic RNRs is a hydroxide ligand on the MnIV.