An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis

Abstract

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2O2, DyPs form a high‐valent iron(IV)‐oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein‐bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein‐bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron‐transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron‐transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

Serial Femtosecond Zero Dose Crystallography Captures a Water‐Free Distal Heme Site in a Dye‐Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV=O Formation

“… The SFX heme site structure of Fe III ‐DtpB and comparison with the redox state validated Fe III ‐DtpA [3d] and Fe III ‐DtpAa [5b] structures. Water molecules (w) are depicted as cyan spheres, hp refers to heme propionate groups and H‐bond interactions are shown as dashed lines.…”

“…The heme‐Fe in DtpB is penta‐coordinate and sits out of the porphyrin plane towards the proximal His225 ligand. Such a distortion results in a short Fe III ‐N δ ‐His bond length (2.05±0.13 Å in monomer A, Table S3) compared to 2.19 Å reported in Fe III ‐DtpA and Fe III ‐DtpAa [3d,e, 5b] . The N ϵ of His225 is H‐bonded to the O δ2 atom of Asp287, an interaction that imparts significant imidazolate character in histidine ligated heme peroxidases resulting in increased electron‐donating ability [2c] .…”

“…[2d] However,amajor complication has been the sensitivity of the heme-Fet or eduction caused by the incident X-rays and lasers. [2d] X-rays interact with protein crystals to generate large numbers of solvated photoelectrons,a nd the effects manifest first in electron rich sites such as metals to rapidly change,for example,the electronic state of the heme-Fea nd with it the structure of the active site.Alternative approaches have been sought to obtain intact (undamaged) or near-intact structural data of radiation-sensitive Fe III and Fe IV heme species.T hese include low-dose composite (i.e.multi-crystal) X-ray structures coupled with single crystal spectroscopy (near-intact), [3] as well as neutron diffraction [4] and structures determined by X-ray free electron lasers (XFELs,z ero dose) [5] to obtain fully intact structures.…”

“…Validated redox‐state structures for DyPs have only been reported for two A‐type homologues from Streptomyces lividans , [3d,e, 5b, 15] providing much needed structural clarity into deciphering the mechanistic features associated with compound I formation in the DyP family [3e] . Significantly, a distal H 2 O‐Asp unit (analogous to the H 2 O‐His unit in nonmammalian peroxidases) was found and experimentally demonstrated to facilitate rapid proton transfer for compound I formation [3e] .…”

Serial Femtosecond Zero Dose Crystallography Captures a Water‐Free Distal Heme Site in a Dye‐Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV=O Formation

“…The N ϵ of His225 is H‐bonded to the O δ2 atom of Asp287, an interaction that imparts significant imidazolate character in histidine ligated heme peroxidases resulting in increased electron‐donating ability [2c] . Fe III ‐DtpA and Fe III ‐DtpAa have a hexa‐coordination heme geometry with a H 2 O molecule occupying the distal coordination position, which forms the origin for an extensive H‐bonded H 2 O network connecting the distal heme‐Fe to bulk solvent [3d] . This H 2 O network communicates with the distal Asp (Figure 2), which in DtpA is optimized for rapid proton movement and compound I formation [3e] .…”