The Histone H3-H4 Tetramer is a Copper Reductase Enzyme

Abstract

The eukaryotic histone H3-H4 tetramer contains a putative Cu²⁺ binding site at the interface of the apposing H3 proteins with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus H3-H4 tetramer is an oxidoreductase enzyme that binds Cu²⁺ and catalyzes its reduction to Cu¹⁺ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu¹⁺ levels and copper-dependent mitochondrial respiration and Sod1 function in the yeast S. cerevisiae. The histone H3-H4 tetramer, therefore, constitutes a novel mechanism for generation of biousable Cu¹⁺ ions in eukaryotes.

One Sentence Summary:

The histone H3-H4 tetramer catalyzes cupric ion reduction to support cellular copper utilization.

Eukaryotes owe their nucleosomal chromatin structure to ancestral histone-containing archaea. Archaeal histones form a structure similar to the eukaryotic H3-H4 tetramer (1) but unlike eukaryotic histones, typically lack extended N-terminal tails and post-translational modifications. Archaea also have smaller genomes than eukaryotes, and no nuclei. The little apparent capability for epigenetic regulation or need for genome compaction in archaea prompted us to consider whether the conserved histone H3-H4 tetramer may have an additional function.

An overlooked feature of the nucleosome and the geochemical events surrounding eukaryogenesis hinted at what such a function might be. The dimerization interface of the two histone H3 proteins contains cysteine and histidine residues (Fig. 1A) in an arrangement typical of Cu²⁺ binding sites in other proteins (2). The evolutionary conservation of residues in this region is greater than expected from their contributions to nucleosome stability (3), consistent with a potential metal binding capability (4–6).

Figure. 1. Recombinant X. laevis histone H3-H4 tetramer interacts with cupric ions.

(A) Left: X. laevis (Xl) nucleosome core particle structure (PDB:1KX5) (38). The box delineates the H3-H3’ interface. Right: Interface residues H3H113 and H3C110 are shown. (B) Alignment of the C-terminal region of S. cerevisiae and H. sapiens histone H3 and archaeal (M. fervidus) histones. (C) Left: UV-vis absorbance spectrum of the Xl H3-H4 tetramer incubated with or without Cu²⁺. Inset: Differential absorbance compared to tetramer without Cu²⁺. Right: Buffer-corrected differential absorbance of the indicated Xl tetramers. (D) Representative ITC titration profile of the Xl H3-H4 tetramer. Circles are experimental data and the line is the fitted curve. Average dissociation constant, enthalpy change, and stoichiometry (N) ±SD of the H3-H4 tetramer-Cu²⁺ complex calculated from three experiments are shown.

The emergence of eukaryotes coincided approximately with the initial accumulation of oxygen (7), which decreased the biousability and increased the toxicity of transition metals such as copper (8). The hypothesis that histones may have facilitated metal utilization in oxidizing conditions has not been considered.

Copper often serves as a redox co-factor for enzymes such as cytochrome c oxidase in the mitochondrial electron transport chain (ETC) and Cu, Zn-superoxide dismutases (e.g., Sod1) (9). As a redox co-factor, copper cycles between the cupric (Cu²⁺) and cuprous (Cu¹⁺) states. However, it is the cuprous form that is trafficked and sensed intracellularly, suggesting a need to maintain cellular copper in the Cu¹⁺ oxidation state for proper distribution and utilization (10). Whether protein-based mechanisms have evolved to regulate intracellular Cu²⁺ reduction is unknown. Here, we present evidence that the eukaryotic H3-H4 tetramer binds Cu²⁺, catalyzes Cu²⁺ reduction and improves the utilization of copper by nuclear, cytoplasmic and mitochondrial proteins.

Recombinant histone H3-H4 tetramer binds cupric ions

To determine whether copper ions interact with the residues at the H3-H3’ interface (4) (Fig. 1A–B), we assembled and purified recombinant Xenopus laevis wildtype (Xl WT) histone H3-H4 tetramers (11) (fig. S1A–C). Incubation of the Xl WT tetramer with increasing amounts of Cu²⁺ in a Tricine buffer resulted in a UV absorbance band that is characteristic of copper-cysteine interactions (12) (Fig. 1C, fig. S1D). UV absorbance plateaued at a molar ratio of ~1, suggesting a stoichiometry of one copper ion per tetramer (Fig. 1C, fig. S1D). Mutation of C110 to alanine (H3C110A) (fig. S1B–C) abolished this copper-dependent absorbance change (Fig. 1C). Isothermal Titration Calorimetry (ITC) analysis further demonstrated high affinity of the tetramer for Cu²⁺ and an approximately equimolar stoichiometry (Fig. 1D). The WT H3-H4 complex also exhibited greater retention on a Cu²⁺ affinity column than the H3^C110A counterpart (fig. S1E).

The histone H3-H4 tetramer catalyzes cupric ion reduction

We next hypothesized that a mechanism to maintain the Cu¹⁺ state intracellularly may be beneficial in oxidizing conditions. Thus, we asked whether the H3-H4 tetramer catalyzes the reduction of Cu²⁺ to Cu¹⁺ in the presence of a source of electrons. We developed an assay utilizing chelators neocuproine (NC) or bicinchoninic acid (BCA) to detect Cu¹⁺ production spectrophotometrically (fig. S2A, Fig. 2A). Assays contained reduced forms of either tris(2-carboxyethyl)phosphine (TCEP), nicotinamide adenine dinucleotide (NADH), or its phosphate form (NADPH), as electron donors. Reactions were initiated by addition of Cu²⁺ in the form of either CuCl₂-Tricine, CuCl₂-N-(2-Acetamido)iminodiacetic acid (ADA), or CuCl₂-Nitrilotriacetic acid (NTA) solutions (fig. S2B). Spontaneous Cu¹⁺ production occurred at a slow rate, but the rate of Cu¹⁺ production substantially increased in the presence of the Xl WT tetramer (Fig. 2B, fig. S2C–E). Production of Cu¹⁺ eventually plateaued due to near full consumption of the electron source (Fig. 2B). No significant production of Cu¹⁺ occurred in the absence of TCEP, indicating that it, and not the tetramer, was the source of electrons. The tetramer enhanced Cu²⁺ reduction in anaerobic conditions as well, ruling out any role for oxygen (fig. S2F). The Xl WT tetramer assembled into a complex with the C-terminus of the human histone chaperone Spt2 (13) also enhanced Cu²⁺ reduction (fig. S2G–I). Additionally, the tetramer could utilize natural reductants such as NADPH to increase Cu²⁺ reduction (fig. S3A). Direct measurement of NADPH confirmed its oxidation during Cu²⁺ reduction (fig. S3B). The effect of the tetramer was specific to cupric ions as there was no enhancement of ferric ion reduction.

Figure. 2. The Xenopus laevis H3-H4 tetramer catalyzes reduction of cupric ions.

(A) Photographic representation of in vitro Cu²⁺ reduction assay. The yellow color is due to NC₂-Cu¹⁺ complex formation. (B) Progress curves of Cu²⁺ reduction with 1 μM of tetramer, heated tetramer, or buffer in presence of 30 μM TCEP and 1 mM CuCl₂-10 mM Tricine. Lines and shading represent the mean ±SD of 3–5 assays. (C-F) Same as (B) but with the indicated amount of (C) TCEP or (D) CuCl₂; or with (F) 5 μM of tetramers in presence of 100 μM TCEP and 0.5 mM CuCl₂-ADA pH 8.

Heating (Fig. 2B) or Proteinase K digestion (fig. S3C) of the Xl WT tetramer abolished Cu²⁺ reduction activity. The unassembled Xl histone H3 or the yeast H2A also did not enhance Cu²⁺ reduction (fig. S3C–D). Increasing rates of Cu¹⁺ production occurred with increasing amounts of either TCEP (Fig. 2C) or Cu²⁺ (Fig. 2D), eventually approaching a maximum rate, consistent with protein-based catalysis (fig. S3E–F).

The H3C110A mutation substantially reduced the rate of Cu²⁺ reduction (Fig. 2E), as did treatment with N-ethylmaleimide (NEM) (fig. S3G). RNase A, which contains redox reactive cysteines (14), did not enhance Cu²⁺ reduction (fig. S3H). Similarly, DTT directly reduced an equimolar amount of Cu²⁺ but did not substantially enhance the rate of Cu²⁺ reduction by TCEP (fig. S3H). These data underscore the importance of H3C110 to the tetramer enzyme activity.

Mutation of H3H113 (4, 5) to alanine (H3H113A), or to asparagine or tyrosine (H3H113N or H3H113Y) which have been found in certain cancers (15), had little effect on the rate of reduction with Cu²⁺-Tricine as the substrate but diminished Cu²⁺ reduction with Cu²⁺-ADA (Fig. 2F, fig. S2B) or Cu²⁺-NTA as substrates (fig. S2B, fig. S3I), likely due to the differing Cu²⁺ coordination ability of different buffers. Altogether, our data reveal that the H3-H4 tetramer catalyzes Cu²⁺ reduction by various electron donors and is thus a cupric reductase.

The yeast histone H3 contains H113 but lacks the C110 residue found in most other eukaryotic H3s (Fig. 1B). Consistently, recombinant yH3-H4 tetramer did not absorb UV light in the 245 nm range when incubated with Cu²⁺. However, a broad peak centered at 680 nm was observed at high concentrations of yeast tetramer and high ionic strength (2 M NaCl), consistent with weakly-absorbing d-d transitions typical of coordinated Cu²⁺ ions (16) (Fig. 3A, fig. S4A). The H3H113A mutation (fig. S4B–C) decreased the copper-dependent absorbance change at 680 nm (Fig. 3A). Furthermore, retention of the yH3-H4 complex on a Cu²⁺ affinity column was reduced by the H3H113A, N, or Y mutations (fig. S4B–F), consistent with a role of H3H113 in Cu²⁺ interaction.

Figure. 3. The yeast H3-H4 tetramer potentially is a copper reductase.

(A-B) Buffer-corrected differential absorbance of the indicated yeast tetramers. (C) Progress curves of Cu²⁺ reduction with 5 μM of tetramers in presence of 100 μM TCEP and 0.5 mM CuCl₂-ADA pH 8.

Mutation of H3A110 to cysteine (yH3^A110C tetramer) (fig. S4G–H) elicited a copper-dependent UV absorbance band nearly identical to that observed with the Xl WT tetramer (Fig. 3B). Furthermore, the H3H113A mutation of the yH3^A110C tetramer (i.e. yH3^A110CH113A tetramer) (fig. S4G–I) diminished copper-dependent absorbance at 245 nm (Fig. 3B), indicating that H3H113 affects the cysteine-Cu²⁺ interaction. The yH3^A110C tetramer also displayed cupric reductase activity, which was diminished by the H3H113A mutation (Fig. 3C). In the absence of H3C110, the yeast tetramer did not display cupric reductase activity in standard salinity, likely due to deficient Cu²⁺ binding, which required 2 M NaCl (Fig. 3A). High salinity interfered with other assay components, precluding further investigation. Nonetheless, our findings suggest that the yH3-H4 tetramer can display cupric reductase activity, and that H3H113 participates in this function.

Mutation of histone H3H113 results in loss of function in yeast

We next investigated whether mutation of the H3H113 residue in vivo would diminish the Cu¹⁺ pool. Mutation of H3H113 to alanine is lethal in S. cerevisiae (17) (fig. S5A). However, introduction of either H3H113N or H3H113Y mutations in the two chromosomal copies of the histone H3 gene generated viable and somewhat slow growing yeast strains H3^H113N and H3^H113Y (fig. S5B). Yeast strains in which the H3H113N and H3H113Y mutant histones were expressed from one locus, with the other locus deleted, were severely sick compared to strains expressing both histone loci (fig. S5C). This finding identifies H3H113N and H3H113Y as loss-of-function alleles, coincident with the loss of Cu²⁺ binding in vitro. We mostly utilized the H3H113N mutation in genetic experiments because H3H113Y results in a more severe growth defect, potentially confounding the interpretation of phenotypes.

H3H113 mutations alter cuprous ion availability

We next assayed the activity of the transcription factor Mac1 as a readout of nuclear Cu¹⁺ abundance because it is directly inhibited by Cu¹⁺ and does not bind Cu²⁺ (18). Gene expression analysis revealed largely similar profiles in WT and H3^H113N strains in rich fermentative media (SC and YPD) (fig. S6A). However, Mac1 target genes were upregulated in both the H3^H113N (fig. S6B) and H3^H113Y strains (Fig. 4A). Deletion of CTR1, the main copper importer in yeast (19), expectedly increased Mac1 target expression (Fig. 4B, fig. S6B–C). However, Mac1 targets displayed even greater upregulation and incomplete repression in response to exogenous copper in H3^H113Nctr1Δ (Fig. 4B, fig. S6C). Importantly, ctr1Δ substantially reduced total copper levels whereas the H3H113N mutation did not (Fig. 4C), suggesting that the H3H113 mutations decrease Cu¹⁺ levels without affecting total copper abundance.

Figure. 4. H3H113 regulates Cu¹⁺-dependent transcriptional activities of Mac1 and Cup2.

(A-B) RT-qPCR analyses of Mac1 target gene (39) expression relative to WT and normalized to ACT1 expression. Boxes (A) and bars (B) show means from four independent experiments (dots) in YPD ±CuSO₄. Baseline copper concentration in YPD is ~1 μM. (C) Intracellular copper content of exponentially growing strains. Bars are means ±SD from 3–6 experiments. (D) Schematic representation of the p^(CUP1)-GFP reporter system. (E-F) Average flow cytometry distributions of cells containing the p^(CUP1)-GFP plasmid grown in SC-ura ±BCS or CuSO₄ from 5–6 experiments. Baseline copper concentration in SC is ~0.25 μM. ***P≤0.001.

We developed a reporter plasmid to assess Cu¹⁺ abundance by employing the Cup2 transcription factor, which is directly activated by Cu¹⁺ but not Cu²⁺ (20). The plasmid harbors the GFP gene downstream of the CUP1 promoter, which is the main target of Cup2 (21) (Fig. 4D, fig. S6D). GFP expression was proportional to the expected Cu¹⁺ levels in both physiological and genetic assays (Fig. 4E, fig. S6E–F), and was reduced in H3^H113N (Fig. 4F, fig. S6G), further indicating that H3H113 is required to maintain cellular Cu¹⁺ levels.

The H3H113 residue supports copper utilization for mitochondrial respiration

We next asked whether the H3H113 mutations impair copper utilization outside the nucleus. We first tested mitochondrial respiration via copper-dependent cytochrome c oxidase function. Although H3^H113N did not display a defect in mitochondrial respiration, the H3^H113Y strain displayed a significant loss of O₂ consumption (Fig. 5A, fig. S7A). This defect was exacerbated in presence of the copper chelator bathocuproinedisulfonic acid (BCS) and was recovered by excess exogenous copper (fig. S7B–C).

Figure. 5. H3H113 is required for utilization of copper for mitochondrial respiration and Sod1 function.

(A-B) Oxygen consumption assays of cells incubated for 18 hrs (A) or 4 hrs (B) in liquid YPEG ±CuSO₄. Baseline copper concentration in YPEG is ~1 μM. Bars show means in linear (A) and log₂ (B) scale ±SD from three experiments. Bars in (A) are scaled to mitochondrial DNA contents. (C) Cytochrome c oxidase assays of cells incubated for 4 hrs in liquid YPEG ±CuSO₄. Bars show means in log₂ scale ±SD from three experiments. N.D.: not detectable. (D) Spot test assays in media ±CuSO₄. (E) Intracellular copper content of cells grown in YPEG ±CuSO₄. Bars show means ±SD from 3–6 experiments. ^#The ctr1Δ strains, which do not grow in non-fermentable media, were incubated in YPEG for 12 hrs and assessed for metal content for reference. (F) Representative Sod1 activity (top) and Sod1 disulfide bond assays (bottom) from three experiments for cells grown in SC ±CuSO₄. Relative signal intensities are indicated (bottom numbers are the ratio of oxidized to total Sod1). (G) Same as (F), except for cells grown in minimal medium ±CuSO₄. Baseline copper concentration in minimal medium is ~0.25 μM. *P≤0.05, **P≤0.01.

To relate the defects in mitochondrial respiration to disruption of copper utilization, we assessed the ability of cells to increase respiratory function as they were shifted from a copper-depleted state to a copper-replete state. Copper depletion via CTR1 deletion resulted in a low rate of antimycin-sensitive O₂ consumption (Fig. 5B, fig. S7D) and cytochrome c oxidase activity (Fig. 5C), which were gradually increased by addition of exogenous copper. However, substantially more copper was required to rescue ctr1Δ in the context of H3H113N (Fig. 5B–C). More copper was also required to rescue respiratory growth of ctr1Δ in the context of H3H113N or H3H113Y (Fig. 5D, fig. S7E). H3H113N also diminished the copper-dependent rescue of cells lacking MAC1, which activates CTR1 expression (18) (fig. S7F). Addition of iron, zinc, or manganese did not rescue the respiratory growth defects of ctr1Δ strains (fig. S7G).

Consistent with the hypomorphic nature of H3H113N, a strain with one WT H3 and one H3H113N gene displayed an intermediate defect compared to strains containing two H3H113N or two WT H3 genes (fig. S8A). Similarly, deleting one of the two copies of the H3 and H4 genes (hht1-hhf1Δ) (fig. S8B–D) increased the copper requirement in ctr1Δ for respiratory growth (fig. S8E).

Total copper and iron levels were similar between H3^H113N and WT in respiratory medium (Fig. 5E, fig. S9A). Addition of excess copper increased total copper levels similarly in both strains (Fig. 5E), indicating that the copper utilization defect in H3^H113Nctr1Δ was not due to changes in total copper abundance. Inefficient recovery of ctr1Δ in the context of H3H113N was not due to increased Cu¹⁺ sequestration by the metallothionein Cup1 because loss of Cup1 (cup1^F8stop) had no effect on the requirement for copper in H3^H113Nctr1Δ (fig. S9B).

We considered whether potential disruptions in chromatin accessibility or gene regulation account for the copper utilization defects of the H3H113 mutants. The H3H113N mutation or deletion of Asf1, a histone chaperone that facilitates nucleosome assembly in part through interaction with H3H113 (22, 23), resulted in minimal disruption of chromatin accessbility (fig. S9C). However, asf1Δ did not phenocopy the H3H113 mutants in respiratory media (fig. S9D). Global gene expression patterns were similar between WT and H3^H113N strains in respiratory media (fig. S9E), with comparable upregulation of genes involved in respiratory growth and copper regulation (fig. S9F–G).

Deletion of membrane-bound metalloreductases FRE1 and FRE2 (24) did not phenocopy the H3H113 mutations (fig. S10A), suggesting that the intracellular role of histone H3 on copper utilization is distinct from extracellular metal reduction. Glutathione (GSH) also participates in cellular copper metabolism (25). Decreasing GSH levels, by deleting the GSH1 gene, increased the amount of copper required to rescue ctr1Δ but to a lesser extent than H3H113N (fig. S10B). Importantly, combining H3H113N with gsh1Δ caused an even greater defect in copper utilization (fig. S10B), suggesting that histones have a unique role in copper utilization.

Histone H3 supports copper utilization for Sod1 function.

We next considered whether histone H3 might also affect copper utilization by Sod1. Total Sod1 activity was ~40% less in H3^H113Y compared to WT, which was recovered by excess exogenous copper (fig. S11A–B). H3H113N also decreased Sod1 activity by ~20% (Fig. 5F). Deletion of CTR1 reduced Sod1 activity and formation of its internal disulfide bond, which are dependent on Cu¹⁺ and the copper chaperone Ccs1 (26) (Fig. 5F). Combining ctr1Δ with H3H113N further decreased the internal disulfide bond and Sod1 activity, which were restored by addition of excess exogenous copper (Fig. 5F). Loss of function of Sod1 causes lysine auxotrophy in yeast (27). Correspondingly, H3^H113Nctr1Δ exhibited a growth defect in lysine deficient conditions (fig. S11C).

Deletion of CCS1 significantly decreased Sod1 activity (Fig. 5G), which was restored by addition of excess copper, but the H3H113N mutation substantially increased the amount of copper required for recovery (Fig. 5G). Considerably more copper was also required to rescue the lysine auxotrophy of ccs1Δ in the context of H3H113N (fig. S11D–E). Unlike the effect of H3H113N, deletion of CTR1 did not increase the requirement of ccs1Δ for exogenous copper (fig. S11F), suggesting that histones contribute to copper utilization differently than Ctr1. Excess copper did not rescue the lysine auxotrophy of sod1Δ strains (fig. S11G), whereas hypoxia rescued the lysine auxotrophy of ccs1Δ and sod1Δ regardless of H3H113 (fig. S11H), indicating that the H3H113 is relevant to this phenotype when Sod1 function is required.

Addition of manganese, zinc, or iron did not rescue ccs1Δ lysine auxotrophy (fig. S11I). The increased copper requirement of H3^H113Nccs1Δ was not due to differences in Cup1-dependent Cu¹⁺ sequestration (fig. S12A), intracellular copper and iron levels (fig. S12B–C) or Asf1 (fig. S11E). Gene expression differences also did not account for defective copper utilization as ccs1Δ and H3^H113Nccs1Δ displayed similar patterns (fig. S12D–F). Altogether, our findings reveal that the H3H113 residue is also important for copper utilization by Sod1.

The significant impacts of H3H113 mutations on Cu¹⁺ abundance and on at least three separate copper-dependent functions support the model that the cupric reductase function of the H3-H4 tetramer regulates Cu¹⁺ availability in yeast.

The H3A110C mutation enhances cuprous ion availability and copper utilization.

The in vitro copper reductase results suggested that H3A110C might serve as a gain-of-function mutation to increase Cu¹⁺ and improve copper utilization in vivo. The yeast H3^A110C strain grew similarly to WT cells (fig. S13A) but enhanced the ability of exogenous copper to restore the respiratory growth of ctr1Δ (Fig. 6A). We reasoned that increased provision of Cu¹⁺ might mitigate the depletion of cellular GSH. Indeed, H3^A110Cgsh1Δ grew better than gsh1Δ in copper-depleted respiratory conditions (fig. S13B) with a greater O₂ consumption rate (Fig. 6B, fig. S13C). H3^A110C exhibited a copper-dependent growth advantage in the presence of a sub-lethal amount of potassium cyanide, an inhibitor of cytochrome c oxidase, independently of GSH levels (Fig. 6C, fig. S13D). Correspondingly, the H3^A110Cgsh1Δ strain displayed greater Cup2 activity than gsh1Δ (Fig. 6D), indicating increased Cu¹⁺ abundance. Additionally, the H3A110C mutation enhanced the copper-dependent rescue of lysine auxotrophy in ccs1Δ (Fig. 6E). The H3A110C mutation even rescued the diminished Cup2 activity of H3^H113N (fig. S13E), the copper utilization defect of the H3^H113Nctr1Δ strain (Fig. 6F), and remarkably, the lethality of the H3H113A mutation (Fig. 6G), corroborating the opposing effects of the H3A110 and H3H113 mutations both in vivo and in vitro. These results highlight the advantage in copper utilization conferred by the H3A110C mutation and further support that the yeast histone H3 regulates the Cu¹⁺ pool and copper utilization.

Figure. 6. The H3A110C mutation enhances copper utilization in S. cerevisiae.

(A) Growth after 48 hrs in liquid YPEG ±CuSO₄. Bars show mean OD₆₀₀ ±SD from three experiments. (B) Oxygen consumption assays of cells grown in the indicated media for 12 hrs. Bars show mean ±SD of three experiments and are scaled to mitochondrial DNA contents. (C) Growth curves in YPEG +potassium cyanide (KCN) ±CuSO₄. Lines show means at each time point ±SD from three experiments. P-value is based on all time points. (D) Average flow cytometry distributions of cells containing the p^(CUP1)-GFP plasmid grown in liquid media ±CuSO₄ from three experiments. (E) Same as (C) but for cells grown in SC-lys ±CuSO₄ from four experiments. (F) Spot test assays in media ±CuSO₄. (G) Plasmid shuffle assay with strains harboring WT H3 on a URA3 plasmid and the indicated H3 gene on a TRP1 plasmid. *P<0.05, **P≤0.01, ***P≤0.001.

Discussion

Histones have been known as packaging and regulatory proteins for the eukaryotic genome. We now reveal that the histone H3-H4 tetramer is also a cupric reductase that provides biousable Cu¹⁺ for the cell. The enzymatic activity suggests the protein complex has many uncharacterized features, including a catalytic site at the H3-H3’ interface, that contribute to Cu²⁺ binding, increasing its reduction potential and promoting electron transfer (28).

The H3-H3’ interface forms in vivo mostly during nucleosome assembly. Therefore, the commencement of enzymatic activity may be coupled to the protection of DNA as it wraps around the nucleosome. Such a coupling may decrease the toxicity of Cu¹⁺ by the very enzymes that produce it as a beneficial adaptation in species that require its intracellular use. However, proteins such as Spt2 (13), may stabilize the H3-H3’ interface outside of the nucleosomal context, affecting regulation and timing of enzymatic activity relative to other chromatin-based events.

The mechanism of electron transfer by the tetramer is unclear but may involve direct transfer from reductants to Cu²⁺ at the active site or through a chain of residues from a distant site (29, 30). Copper is present in the nucleus (31, 32), but is likely bound (33) and transported to and from histones through as-yet-to-be-identified chaperones, possibly including Atox1 (34). Interestingly, copper disproportionately accumulates in the nucleus when its cellular efflux is compromised such as in Wilson’s disease (35, 36), suggesting that the nucleus is a major transit hub for cellular copper.

The oxidoreductase function of the H3-H4 tetramer provides a reasonable hypothesis for why the archaeal ancestor of the eukaryotes possessed histone tetramers. Such enzymatic activity, with an associated intracellular Cu¹⁺ transit system, could have helped maintain a functioning ETC in the proto-mitochondria, thereby making the presence of histones in the ancestral archaeon not incidental (37), but rather essential for eukaryogenesis.