Self-assembled oligohistidine peptides form crystalline β-sheet nanomaterials whose periodically arranged imidazole side chains catalyse H2O2-driven substrate oxidations without any cofactor, metal centre, or π-conjugated framework, through a proposed surface ternary-complex pathway involving OOH•/O2•− reactive species. The 2021 discovery left the system quantitatively and mechanistically unresolved: the fraction of catalytically accessible histidine residues, and hence the true per-active-site turnover rates, is unknown; the atomistic reaction pathway and its stepwise transition states have not been established; and the structure-activity rules that govern how morphology, facet exposure, and sequence cap the activity are unmapped. The problem asks for the quantitative, structure-resolved mechanistic account of residue-only redox catalysis needed to judge how far such active sites can be pushed toward haem-enzyme performance.

Background

Natural oxidases and peroxidases catalyse electron-transfer reactions with the help of bound cofactors, most commonly haem (an iron-porphyrin complex) held in a precise geometry by the folded protein; horseradish peroxidase (HRP), the workhorse haem peroxidase, reduces hydrogen peroxide (H2O2) while oxidizing substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), and such enzymes are irreversibly inactivated by heat or acid because the cofactor and the supporting fold dissociate or aggregate. Artificial enzyme mimics ('nanozymes') replace the protein with a nanomaterial, but nearly all oxidoreductase-mimicking nanozymes draw their redox chemistry from transition metals (CeO2, Fe3O4, single-atom sites) or from extended π-conjugated carbon frameworks in which charge delocalization is invoked for O2/H2O2 activation. Whether amino-acid side chains alone, stripped of any cofactor and of any extended conjugation, can organize into an active site that performs H2O2/O2 redox catalysis remained an open question until Liu et al. (Nature Materials 20, 395-402, 2021) reported that oligohistidine peptides, from the dipeptide (H2) to the pentadecapeptide (H15), self-assemble in aqueous buffer into β-sheet-based planar nanostructures, with H15 forming single crystals whose histidine imidazole side chains form periodic surface arrays. The imidazole is the five-membered aromatic side-chain ring of histidine, and a β-sheet is an extended, hydrogen-bonded ribbon of peptide chains; here the sheets stack so that imidazole groups line exposed crystal facets.

The H15 assemblies catalyse H2O2-dependent oxidations of TMB, reduced nicotinamide adenine dinucleotide (NADH), and homovanillic acid with Michaelis-Menten saturation kinetics (the standard enzyme-kinetic framework in which the turnover number kcatk_{\mathrm{cat}} counts product molecules formed per catalyst per second and kcat/KMk_{\mathrm{cat}}/K_{\mathrm{M}} reports catalytic efficiency at low substrate concentration). Double-reciprocal plots indicate a ternary-complex mechanism in which TMB and H2O2 are co-adsorbed on the crystal facet (010) before reacting; for this peroxidase-type substrate oxidation, superoxide dismutase suppresses the activity while a hydroxyl-radical scavenger does not, and EPR spin trapping with the BMPO nitrone was taken to identify hydroperoxyl/superoxide (OOH•/O2•−) as the reactive intermediates, whereas separate spin-trapping and scavenger experiments implicate hydroxyl radicals when H2O2 is mixed with H15 and in the catalase-like H2O2 disproportionation. Inductively coupled plasma mass spectrometry and metal-spiking controls argued against trace-metal (Fenton-type) catalysis, and the catalyst could be switched between inactive and active states over ten or more cycles of heating/cooling or acidification/neutralization with full recovery of activity, which the authors presented as a model for primitive, cofactor-free enzymes that could survive prebiotic thermal and pH fluctuations.

Two quantitative limitations were explicit in the source. First, the reported kinetics are normalized to total peptide: for H15, kcatTMB=(1.322±0.050)×10−4 s−1k_{\mathrm{cat}}^{\mathrm{TMB}}=(1.322\pm0.050)\times10^{-4}\ \mathrm{s}^{-1} and kcatTMB/KMTMB=(0.704±0.071) M−1s−1k_{\mathrm{cat}}^{\mathrm{TMB}}/K_{\mathrm{M}}^{\mathrm{TMB}}=(0.704\pm0.071)\ \mathrm{M}^{-1}\mathrm{s}^{-1}, orders of magnitude below cofactor-containing systems, and the authors attribute part of this to the fact that only surface-exposed histidines (for example on facet (010)) are catalytically accessible while the assemblies contain 'a large number of buried active sites' (Supplementary Discussion 4 of the source); the true per-active-site rates are therefore unknown. Second, the mechanistic picture rests on docking-level binding energies of H2O2 and TMB to a powder-XRD-refined structural model plus EPR and kinetics; no stepwise reaction energetics or transition states were computed. Activity enhancements from ultrasonic fragmentation (a roughly 43% gain in initial rate, with the fitted kcatTMBk_{\mathrm{cat}}^{\mathrm{TMB}} rising to (6.21±0.202)×10−4 s−1(6.21\pm0.202)\times10^{-4}\ \mathrm{s}^{-1}) and from conjugating oligohistidine to the fibril-forming Q11 peptide (roughly tenfold, to kcatTMB≈1.6×10−3 s−1k_{\mathrm{cat}}^{\mathrm{TMB}}\approx1.6\times10^{-3}\ \mathrm{s}^{-1}) showed that accessible surface area limits the activity, but well-defined structure-activity relationships for these morphological variables were not established.

Problem Statement

For the cofactor-free oxidase-/peroxidase-mimetic catalysts formed by self-assembled histidine-rich peptides (oligohistidines such as H15 and their fibril-forming conjugates), establish a quantitative, structure-resolved mechanistic account of how ordered imidazole arrays catalyse H2O2-driven substrate oxidation, comprising: (1) the fraction of histidine residues that are catalytically accessible in a given assembly, meaning the residues that function as active sites contributing to the observed turnover rather than the buried residues invoked in the source, and the corresponding true per-active-site kcatk_{\mathrm{cat}} and kcat/KMk_{\mathrm{cat}}/K_{\mathrm{M}} obtained by normalizing the measured rates to that fraction (the peptide-normalized values reported in the source are underestimates because buried residues are inaccessible); (2) the atomistic reaction pathway on the active facet beyond the source's docking-level binding models, including the stepwise energetics and transition states of H2O2 activation and substrate oxidation, which reactive oxygen species operate in each catalytic branch and how they form (the source's branch-wise working assignments, OOH•/O2•− for the peroxidase-type substrate oxidation and •OH for the catalase-like H2O2 disproportionation, are to be established rather than assumed), and how the periodically arrayed, non-conjugated imidazole side chains and their cooperative neighbouring groups mediate electron redistribution, including whether localized aromatic π orbitals participate; and (3) a single general structure-activity relationship governing how particle size, morphology, facet exposure, and sequence control the activity, with the turnover numbers of haem-dependent peroxidases exceeding those of the present cofactor-free systems by three to six orders of magnitude as the motivating open context.

References

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