Metalloenzymes play a critical role in many of the life-giving reactions on the planet, such as photosynthesis and respiration. One group of metalloproteins, the ferritin-like superfamily, is involved in a diverse group of functions ranging from Fe and O₂ storage and transport to C-H bond activation and deoxyribonucleotide biosynthesis. Recently, a new subgroup has been identified within the ferritin-like superfamily that can site-selectively bind both Mn(II) and Fe(II) ions, counter to the well-established Irving-Williams series. This new group has been named the R2-like ligand-binding oxidases, or R2lox, due to its similarity in sequence and metal binding properties to the class 1c R2 subunit of the ribonucleotide reductases. R2lox possesses many characteristics that distinguish it from members of the canonical di-iron ferritin-like proteins, such as the ability to bind either Fe/Fe or Mn/Fe cofactors, the presence of an exogenous fatty acid lipid bound to the metal centers, and the capability to perform two-electron oxidation reactions, as evidenced by formation of a Tyr-Val crosslink upon oxygen activation. Recently, we have observed that R2lox undergoes a structural rearrangement in the active site upon photoirradiation. Using a variety of spectroscopic and photochemical methods, including a state-of-the-art resonance Raman system, a possible structure and mechanism for formation of this new state has been proposed. Future experiments relating this state to a possible biological function will be discussed.

Human PrimPol is a recently discovered bifunctional enzyme that displays template-directed primase activity and DNA polymerase activity. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when coordinating Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. However, the abundance of cellular Mg²⁺ is much greater than that of Mn²⁺. At present, there is debate among those who study PrimPol as to which divalent metal ion cofactor is utilized by this unique protein. We experimentally studied PrimPol in the presence of both cofactors in an attempt to elucidate which of them is preferred. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct nucleotides with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Strikingly, the fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4x10^-2 to 3.8x10^-1, indicating that PrimPol displays extremely unfaithful polymerase activity, which may not be biologically relevant. Additionally, we demonstrated a simple kinetic basis for discrimination by PrimPol to select for deoxyribonucleotides over ribonucleotides with both Mg²⁺ and Mn²⁺ likely arising from decreased nucleotide binding and subsequent incorporation. Furthermore, PrimPol also showed efficient incorporation of the anti-cancer drugs, cytarabine and gemcitabine, onto growing DNA strands revealing its potential role in cellular toxicities induced by these drugs.

DNA absorbs UV radiation strongly, leading to the formation of excited electronic states that can decay to mutagenic photoproducts. In recent years, attention has focused on understanding why excited states in DNA strands are generally much longer-lived than excited states of monomeric nucleobases. Femtosecond transient absorption measurements with mid-infrared probing have shown that UV excitation of single-stranded DNA efficiently forms charge transfer states in which an electron is transferred between π-stacked nucleobases. These states are revealed by vibrational marker bands of nucleobase radical ions, which appear on a subpicosecond time scale and decay by charge recombination in tens to hundreds of picoseconds, depending on base sequence. In double-stranded DNA, intrastrand charge separation produces radical ion base pairs that undergo interstrand proton transfer in some sequences [1]. Proton-coupled electron transfer is complete in less than 200 fs. Recent experiments suggest that the ground state is repopulated in tens of picoseconds via intrastrand back electron transfer followed by much faster interstrand proton transfer [2]. The radicals and tautomeric base pairs formed transiently by UV absorption decay efficiently and do not appear to pose a photochemical threat.

References:
[1] Y. Zhang, K. de La Harpe, A. A. Beckstead, R. Improta, and B. Kohler, J. Am. Chem. Soc. 137, 7059 (2015).
[2] Y. Zhang, K. de La Harpe, A. A. Beckstead, L. Martínez-Fernández, R. Improta, and B. Kohler, J. Phys. Chem. Lett. 7, 950 (2016).