XFEL Crystal Structures of Peroxidase Compound II
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ABSTRACT:
SUBMITTER: Kwon H
PROVIDER: S-EPMC10947387 | biostudies-literature | 2021 May
REPOSITORIES: biostudies-literature
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Project description: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 FeIV =O or FeIV -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 FeIV =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.
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Project description:The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.
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Project description:Structural and spectroscopic investigations of compound II in ascorbate peroxidase (APX) have yielded conflicting conclusions regarding the protonation state of the crucial Fe(IV) intermediate. Neutron diffraction and crystallographic data support an iron(IV)-hydroxo formulation, whereas Mössbauer, X-ray absorption (XAS), and nuclear resonance vibrational spectroscopy (NRVS) studies appear consistent with an iron(IV)-oxo species. Here we examine APX with spectroscopy-oriented QM/MM calculations and extensive exploration of the conformational space for both possible formulations of compound II. We establish that irrespective of variations in the orientation of a vicinal arginine residue and potential reorganization of proximal water molecules and hydrogen bonding, the Fe-O distances for the oxo and hydroxo forms consistently fall within distinct, narrow, and nonoverlapping ranges. The accuracy of geometric parameters is validated by coupled-cluster calculations with the domain-based local pair natural orbital approach, DLPNO-CCSD(T). QM/MM calculations of spectroscopic properties are conducted for all structural variants, encompassing Mössbauer, optical, X-ray absorption, and X-ray emission spectroscopies and NRVS. All spectroscopic observations can be assigned uniquely to an Fe(IV)═O form. A terminal hydroxy group cannot be reconciled with the spectroscopic data. Under no conditions can the Fe(IV)═O distance be sufficiently elongated to approach the crystallographically reported Fe-O distance. The latter is consistent only with a hydroxo species, either Fe(IV) or Fe(III). Our findings strongly support the Fe(IV)═O formulation of APX-II and highlight unresolved discrepancies in the nature of samples used across different experimental studies.
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Project description:The protonation state of the iron(IV) oxo (or ferryl) form of ascorbate peroxidase compound II (APX-II) is a subject of debate. It has been reported that this intermediate is best described as an iron(IV) hydroxide species. Neutron diffraction data obtained from putative APX-II crystals indicate a protonated oxygenic ligand at 1.88 Å from the heme iron. This finding, if correct, would be unprecedented. A basic iron(IV) oxo species has yet to be spectroscopically observed in a histidine-ligated heme enzyme. The importance of ferryl basicity lies in its connection to our fundamental understanding of C-H bond activation. Basic ferryl species have been proposed to facilitate the oxidation of inert C-H bonds, reactions that are unknown for histidine-ligated hemes enzymes. To provide further insight into the protonation status of APX-II, we examined the intermediate using a combination of Mössbauer and X-ray absorption spectroscopies. Our data indicate that APX-II is an iron(IV) oxo species with an Fe-O bond distance of 1.68 Å, a K-edge pre-edge absorption of 18 units, and Mössbauer parameters of ΔEq = 1.65 mm/s and δ = 0.03 mm/s.
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Project description:The protonation state of ascorbate peroxidase compound II (APX-II) has been a subject of debate. A combined X-ray/neutron crystallographic study reported that APX-II is best described as an iron(IV)hydroxide species with an FeO distance of 1.88 Å (Kwon, et al. Nat Commun2016, 7, 13,445), while X-ray absorption spectroscopy (XAS) experiments (utilizing extended X-ray absorption fine structure (EXAFS) and pre-edge analyses) indicate APX-II is an authentic iron(IV)oxo species with an FeO distance 1.68 Å (Ledray, et al. Journal of the American Chemical Society2020,142, 20,419). Previous debates concerning ferryl protonation states have been resolved through the application of Badger's rule, which correlates FeO bond distances with FeO vibrational frequencies. To obtain the required vibrational data, we have collected Nuclear Resonance Vibrational Spectroscopy (NRVS) data for APX-II. We observe a broad vibrational feature in the range associated with iron(IV)oxo stretching (700-800 cm-1). This feature appears to have two peaks at 732 cm-1 and 770 cm-1, corresponding to FeO distances of 1.69 and 1.67 Å, respectively. The broad vibrational envelope and the presence of multiple resonances could reflect a distribution of hydrogen bonding interactions within the active-site pocket.
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Project description:The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
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