Charge Transfer between [4Fe4S] Proteins and DNA Is Unidirectional: Implications for Biomolecular Signaling

Abstract

Recent experiments suggest that DNA-mediated charge transport might enable signaling between the [4Fe4S] clusters in the C-terminal domains of human DNA primase and polymerase α, as well as the signaling between other replication and repair high-potential [4Fe4S] proteins. Our theoretical study demonstrates that the redox signaling cannot be accomplished exclusively by DNA-mediated charge transport because part of the charge transfer chain has an unfavorable free energy profile. We show that hole or excess electron transfer between a [4Fe4S] cluster and a nucleic acid duplex through a protein medium can occur within microseconds in one direction, while it is kinetically hindered in the opposite direction. We present a set of signaling mechanisms that may occur with the assistance of oxidants or reductants, using the allowed charge transfer processes. These mechanisms would enable the coordinated action of [4Fe4S] proteins on DNA, engaging the [4Fe4S] oxidation state dependence of the protein-DNA binding affinity.

Figure 1.

Human primosome-nucleic acid complex. (A) Portion of the primosome crystal structure (PDB ID 5EXR) highlighting the [4Fe4S] cluster docked to p180c (at zinc binding site 1, ZBS1) relative to p58c [4Fe4S] cluster and to the other zinc binding site (ZBS2) in p180c. Color code: zinc (red), S (yellow), Fe (magenta), p180core (cyan), p180c (orange), p58c (silver), p58N (pink), p180core-p180c linker (green), p58c-p58N linker (blue). (B) Schematic view of the protein complex bound to RNA/DNA. p58c, with an oxidized [4Fe4S] cluster (yellow), is linked to primase N-terminal domain (p58N). p58N connects primase to Polα. We show the catalytic core (p180core) linked to the C-terminal subunit p180c of Polα, and a [4Fe4S] cluster (orange) bound to p180c. The nucleic acid transiently associates primase and Polα. The distance between the p180c [4Fe4S] cluster and the duplex depends on the conformation of the p180core-p180c linker. The p58c [4Fe4S] cluster is at edge-to-edge distances of 50.9 Å and 38.6 Å from ZBS2 and from the [4Fe4S] cluster docked to p180c, respectively. For hole transport exclusively mediated by the duplex, the path of the charge from one [4Fe4S] cluster to the other would consist of: step 1_h from the initially oxidized iron-sulfur cluster in p58c (oxidation state 3+, represented as a yellow cube) to the duplex, through the protein medium; step 2_h, namely, the injected hole, h⁺, moves across the duplex; step 3_h from the duplex to the cluster in p180c, which may occur with or without protein mediation depending on the protein arrangement in Polα and the positioning of the duplex. In the case of excess electron transport, the CT path would be as follows: the initially reduced p180c cluster (oxidation state 2+, represented as on orange cube) injects an electron, e⁻, into the initially neutral duplex; the excess electron transfers to the nucleic acid portion close to Polα (step 2_e); the electron is donated from the anionic duplex to p58c (step 3_e).

Figure 2.

CT-mediated [4Fe4S] protein signaling and pertinent redox potentials. (A) Possible mechanisms for protein communication via CT, supported by the primer: 1) hole transfer or 2) excess electron transfer between the p58c and p180c [4Fe4S] clusters (drawn as small cubes), mediated by a RNA/DNA duplex transiently bound to both p58c and p180core. Part of the CT route (in red) is energetically unfavorable. Nearby oxidants (O) or reductants (R) should support these signaling mechanisms. 3) Change in relative DNA-binding affinities of primase and Polα caused by direct inter­cluster CT. 4) Competitive protein binding to the primer, modulated by sequential changes in the [4Fe4S] cluster oxidation states assisted by RNA/DNA and surrounding redox agents. (B) Redox potential landscape (Table S1) for hole transfer (blue) and excess electron transfer (green), and related downhill (a, c) and uphill (b, d) electron transfer processes. Mechanism 1 would first require hole hopping from [4Fe4S] ³⁺in p58c to the duplex through protein redox-active residues. However, the hole transfer to any amino acid (that is, the electron-transfer step b in the opposite direction) is energetically uphill, thus making the hole hopping process kinetically unfeasible (forbidden step in mechanism 1). If the duplex is oxidized by an external agent (O), the hole can be delivered to [4Fe4S]²⁺ in p180c, that is, an electron can transfer downhill in the opposite direction (step a), from the [4Fe4S]²⁺ energy level to one of the duplex energy levels, possibly occupying intermediate Tyr and Trp levels. Mechanism 2 would first require direct or protein-mediated electron transfer from [4Fe4S]²⁺ in p180c to the duplex (uphill step d, that is, kinetically unfeasible step in mechanism 2). However, if the duplex is negatively charged by an external agent (R), the excess electron can move downhill from any of the purine energy levels, through Tyr and Trp levels, to reduce the initially oxidized p58 cluster (allowed electron transfer step c). The dashed blue lines correspond to different choices for Tyr and Trp oxidation potentials that are described in the main text.

Figure 3.

CT steps and rate constants (in s⁻¹) in hole hopping between the iron-sulfur cluster and the nucleobases in the p58c-RNA/DNA complex (crystal structure with PDB ID 5F0Q.) The CT steps with an inverse rate constant within a biologically relevant millisecond time scale are in blue; the other steps are in red. The fastest (second fastest) CT route is drawn as a green (orange) dashed line. Possible routes for charge transport between the [4Fe4S] cluster and the nucleic acid are 1: [4Fe4S]-M307-DA7; 2: [4Fe4S]-Y309-W327-M307-DA7; 3: [4Fe4S]-Y309-W327-Y345-GTP; 4: [4Fe4S]-Y309-W327-Y345-Y347-DG4; 5: [4Fe4S]-M288-Y309-W327-M307-DA7; 6: [4Fe4S]-M288-Y309-W327-Y345-GTP; 7: [4Fe4S]-M288-Y309-W327-Y345-Y347-DG4.

Figure 4.

Kinetic model for electron transfer from the [4Fe4S] cluster in p58c to the anchored nucleic acid. 0 is the iron-sulfur cluster; 1 to N denote the primase redox-active residues that intervene in a given charge transport pathway; and N + 1 is the electron-accepting purine nucleobase in the nucleic acid. J is the electron charge flux to the nucleic acid and/or other agent in the environment that sweeps away the electron once it arrives at site N + 1 (see main text). Thus, the occupation probability of this site is negligible at any time, and the backward rate k_N+1→N (in gray) does not appear in the corresponding master equation (Eq. S25 of the SI).

Figure 5.

Electron transfer routes from the p58c protein to nucleic acid. The two most rapid electron transfer paths from the p58c [4Fe4S] cluster to RNA/DNA (i.e., hole transfer in the opposite direction) are shown as green ([4Fe4S]-M307-DA7) and orange ([4Fe4S]-Y309-W327-M307-DA7) arrows. The timescale of the second route may range from ms to μs depending on the redox potentials of the Tyr and Trp residues. The purple arrow shows the fastest but unproductive electron transfer from [4Fe4S] to DA10 in the single-stranded DNA portion. H303 (denoted H), which is H-bonded to DA7, and DC6, which is paired to GTP, are in pink.