Peter C. E. Moody

Radiation damage to macromolecular structures remains a significant challenge for accurate structure solution by X-ray crystallography, leading to incorrect structural and chemical interpretation of the data. Site-specific radiation damage is insidious, typically unidentifiable solely from summary statistics, and is primarily discussed with reference to the predominant forms: disulfide-bond cleavage, metal-centre reduction and decarboxylation of acidic residues. A method is presented for identifying potentially oxidatively damaged cysteines by interrogating the accuracy of the built model within the electron density and the geometry of the difference density peaks surrounding a cysteine. We also highlight that cysteines located within protein active sites or that are in hydrolases are predisposed to this damage.

The active sites of heme enzymes have evolved to control the formation of highly reactive intermediates in oxidative catalysis. Proton delivery to the heme is essential, yet the mechanisms of proton delivery remain poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase (ascorbate peroxidase) using computational approaches that combine classical, quantum, and hybrid methods with enhanced sampling and local electric field (LEF) analyses. Our results show that networks of active‐site water molecules facilitate proton exchange with Arg38, which may act as a transient proton carrier at the γ‐heme edge where the substrate binds. The distal His42 residue aids proton transfer into the active site via solvent at the δ‐edge. Molecular dynamics simulations of three heme peroxidases identify hydrated channels leading to both γ‐ and δ‐edges, allowing solvent protons to reach the active site. Comparison with eight other heme peroxidases shows that these channels are conserved. LEF analyses reveal a continuous electrostatic funnel drawing protons toward the heme from the γ‐ and δ‐edges, a feature that is broadly conserved across other peroxidases. These results suggest that nature pre‐organizes electrostatic funnels and solvent channels to provide multiple well‐defined routes for proton delivery in peroxidase catalysis.

The active sites of heme enzymes have evolved to control the formation of highly reactive intermediates in oxidative catalysis. Proton delivery to the heme is essential, yet the mechanisms of proton delivery remain poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase (ascorbate peroxidase) using computational approaches that combine classical, quantum, and hybrid methods with enhanced sampling and local electric field (LEF) analyses. Our results show that networks of active‐site water molecules facilitate proton exchange with Arg38, which may act as a transient proton carrier at the γ‐heme edge where the substrate binds. The distal His42 residue aids proton transfer into the active site via solvent at the δ‐edge. Molecular dynamics simulations of three heme peroxidases identify hydrated channels leading to both γ‐ and δ‐edges, allowing solvent protons to reach the active site. Comparison with eight other heme peroxidases shows that these channels are conserved. LEF analyses reveal a continuous electrostatic funnel drawing protons toward the heme from the γ‐ and δ‐edges, a feature that is broadly conserved across other peroxidases. These results suggest that nature pre‐organizes electrostatic funnels and solvent channels to provide multiple well‐defined routes for proton delivery in peroxidase catalysis.

The active site architecture of heme enzymes has evolved to control the formation of highly reactive intermediates for oxidative catalysis. Finely tuned proton delivery to heme is essential, yet the mechanisms of proton delivery and the sources of protons are poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase enzyme (ascorbate peroxidase) employing a range of computational molecular modeling approaches, including molecular dynamics (MD) simulations, density functional theory (DFT) and combined quantum mechanics/molecular mechanics (QM/MM) calculations, enhanced sampling techniques, and local electric field (LEF) analyses. Our results show that networks of active site water molecules facilitate proton ex-change with Arg38, which may serve as a transient proton carrier at the γ-edge of the heme, where the substrate (ascorbate) binds. The distal His42 residue aids proton transfer into the active site via solvent from the δ-heme edge. MD simulations of three peroxidases identify hydrated channels in the structure – pointing towards both the γ- and δ-edges of the heme – through which protons from solvent can access the active site. Comparison with structures of 8 other heme peroxidases shows that these channels are conserved features. LEF calculations show a continuous electrostatic funnel, running from positive to negative, that pulls protons from the protein surface along the channels towards the heme iron from both the γ- and δ-heme edges. This electric field is also found to be a conserved feature in several peroxidases; its direction and shape is conserved but the gradient varies between differ-ent enzymes, suggesting that nature preorganises the electric field as a determinant of relative activity. Rather than a simplistic model for proton delivery (in which a single substrate provides a single proton (and an electron) to the heme) the data instead show conserved solvent channels and electrostatic funnels, which provide multiple well defined routes for proton delivery in peroxidase catalysis.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme—and whether it is an Fe^IV=O or Fe^IV‐OH species—is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated Fe^IV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X‐ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron‐oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine‐tuning is linked to the functional need for proton delivery to the heme.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme—and whether it is an Fe^IV=O or Fe^IV‐OH species—is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated Fe^IV=O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X‐ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron‐oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine‐tuning is linked to the functional need for proton delivery to the heme.