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A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

ABSTRACT: Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.

SUBMITTER: Ortmayer M

PROVIDER: S-EPMC8317151 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A mononuclear Ni(II) complex having an acireductone type ligand, and supported by the bnpapa (N,N-bis((6-neopentylamino-2-pyridyl)methyl)-N-((2-pyridyl)methyl)amine) ligand, [(bnpapa)Ni(PhC(O)C(OH)C(O)Ph)]ClO(4) (14), has been prepared and characterized by elemental analysis, (1)H NMR, FTIR, and UV-vis. To gain insight into the (1)H NMR features of 14, the air stable analogue complexes [(bnpapa)Ni(CH(3)C(O)CHC(O)CH(3))]ClO(4) (16) and [(bnpapa)Ni(ONHC(O)CH(3))]ClO(4) (17) were prepared and characterized by X-ray crystallography, (1)H NMR, FTIR, UV-vis, mass spectrometry, and solution conductivity measurements. Compounds 16 and 17 are 1:1 electrolyte species in CH(3)CN. (1)H and (2)H NMR studies of 14, 16, and 17 and deuterated analogues revealed that the complexes having six-membered chelate rings for the exogenous ligand (14 and 16) do not have a plane of symmetry within the solvated cation and thus exhibit more complicated (1)H NMR spectra. Compound 17, as well as other simple Ni(II) complexes of the bnpapa ligand (e.g., [(bnpapa)Ni(ClO(4))(CH(3)CN)]ClO(4) (18) and [(bnpapaNi)(2)(mu-Cl)(2)](ClO(4))(2) (19)), exhibit (1)H NMR spectra consistent with the presence of a plane of symmetry within the cation. Treatment of [(bnpapa)Ni(PhC(O)C(OH)C(O)Ph)]ClO(4) (14) with O(2) results in aliphatic carbon-carbon bond cleavage within the acireductone-type ligand and the formation of [(bnpapa)Ni(O(2)CPh)]ClO(4) (9), benzoic acid, benzil, and CO. Use of (18)O(2) in the reaction gives high levels of incorporation (>80%) of one labeled oxygen atom into 9 and benzoic acid. The product mixture and level of (18)O incorporation in this reaction is different than that exhibited by the analogue supported the hydrophobic 6-Ph(2)TPA ligand, [(6-Ph(2)TPA)Ni(PhC(O)C(OH)C(O)Ph)]ClO(4) (2). We propose that this difference is due to variations in the reactivity of bnpapa- and 6-Ph(2)TPA-ligated Ni(II) complexes with triketone and/or peroxide species produced in the reaction pathway.

Project description:Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H(2)S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H(2)S toxicity. Single point mutations at E7, B10, and E11 were introduced into the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild-type recombinant proteins were first evaluated using the well-known ferrous CO and O(2) derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O(2) binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, -PheB10Val, -PheB10Leu, and -E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H(2)S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H(2)S complexes, which show reduction of heme iron as judged by the appearance of the nu(4) oxidation state marker at 1356 cm(-1), indicative of heme-Fe(II) species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting the strongest H-bonding interactions. Overall, the results presented here show that (a) H(2)S association is regulated by external kinetic barriers, (b) H(2)S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction, (c) at high H(2)S concentrations, reduction of the ferric center dominates, and (d) reduction of the heme is also enhanced in those HbI mutants having polar distal environments.

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A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

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