Bioorthogonal Chemistry Enables Single-Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase.

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs' structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

Nonmicrobial Nitrophenol Degradation via Peroxygenase Activity of Dehaloperoxidase-Hemoglobin from Amphitrite ornata.

The marine hemoglobin dehaloperoxidase (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of nitrophenols, an unprecedented nonmicrobial degradation pathway for nitrophenols by a hemoglobin. Using 4-nitrophenol (4-NP) as a representative substrate, the major monooxygenated product was 4-nitrocatechol (4-NC). Isotope labeling studies confirmed that the O atom incorporated was derived exclusively from H2O2, indicative of a peroxygenase mechanism for 4-NP oxidation. Accordingly, X-ray crystal structures of 4-NP (1.87 Å) and 4-NC (1.98 Å) bound to DHP revealed a binding site in close proximity to the heme cofactor. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. The 4-NC product was itself a peroxidase substrate for DHP, leading to the secondary products 5-nitrobenzene-triol and hydroxy-5-nitro-1,2-benzoquinone. DHP was able to react with 2,4-dinitrophenol (2,4-DNP) but was unreactive against 2,4,6-trinitrophenol (2,4,6-TNP). pH dependence studies demonstrated increased reactivity at lower pH for both 4-NP and 2,4-DNP, suggestive of a pH effect that precludes the reaction with 2,4,6-TNP at or near physiological conditions. Stopped-flow UV-visible spectroscopic studies strongly implicate a role for Compound I in the mechanism of 4-NP oxidation. The results demonstrate that there may be a much larger number of nonmicrobial enzymes that are underrepresented when it comes to understanding the degradation of persistent organic pollutants such as nitrophenols in the environment.

Peroxygenase and oxidase activities of dehaloperoxidase-hemoglobin from Amphitrite ornata.

The marine globin dehaloperoxidase-hemoglobin (DHP) from Amphitrite ornata was found to catalyze the H2O2-dependent oxidation of monohaloindoles, a previously unknown class of substrate for DHP. Using 5-Br-indole as a representative substrate, the major monooxygenated products were found to be 5-Br-2-oxindole and 5-Br-3-oxindolenine. Isotope labeling studies confirmed that the oxygen atom incorporated was derived exclusively from H2O2, indicative of a previously unreported peroxygenase activity for DHP. Peroxygenase activity could be initiated from either the ferric or oxyferrous states with equivalent substrate conversion and product distribution. It was found that 5-Br-3-oxindole, a precursor of the product 5-Br-3-oxindolenine, readily reduced the ferric enzyme to the oxyferrous state, demonstrating an unusual product-driven reduction of the enzyme. As such, DHP returns to the globin-active oxyferrous form after peroxygenase activity ceases. Reactivity with 5-Br-3-oxindole in the absence of H2O2 also yielded 5,5'-Br2-indigo above the expected reaction stoichiometry under aerobic conditions, and O2-concentration studies demonstrated dioxygen consumption. Nonenzymatic and anaerobic controls both confirmed the requirements for DHP and molecular oxygen in the catalytic generation of 5,5'-Br2-indigo, and together suggest a newly identified oxidase activity for DHP.

Noncovalent modulation of pH-dependent reactivity of a Mn-salen cofactor in myoglobin with hydrogen peroxide.

To demonstrate protein modulation of metal-cofactor reactivity through noncovalent interactions, pH-dependent sulfoxidation and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) oxidation reactivity of a designed myoglobin (Mb) containing non-native Mn-salen complex (1) was investigated using H2O2 as the oxidant. Incorporation of 1 inside the Mb resulted in an increase in the turnover numbers through exclusion of water from the metal complex and prevention of Mn-salen dimer formation. Interestingly, the presence of protein in itself is not enough to confer the increase activity as mutation of the distal His64 in Mb to Phe to remove hydrogen-bonding interactions resulted in no increase in the turnover numbers, while mutation His64 to Arg, another residue with ability to hydrogen-bond interactions, resulted in an increase in reactivity. These results strongly suggest that the distal ligand His64, through its hydrogen-bonding interaction, plays important roles in enhancing and fine-tuning reactivity of the Mn-salen complex. Nonlinear least-squares fitting of rate versus pH plots demonstrates that 1.Mb(H64X) (X=H, R and F) and the control Mn-salen 1 exhibit pKa values varying from pH 6.4 to 8.3, and that the lower pKa of the distal ligand in 1.Mb(H64X), the higher the reactivity it achieves. Moreover, in addition to the pKa at high pH, 1.Mb displays another pKa at low pH, with pKa of 5.0+/-0.08. A comparison of the effect of different pH on sulfoxidation and ABTS oxidation indicates that, while the intermediate produced at low pH conditions could only perform sulfoxidation, the intermediate at high pH could oxidize both sulfoxides and ABTS. Such a fine-control of reactivity through hydrogen-bonding interactions by the distal ligand to bind, orient and activate H2O2 is very important for designing artificial enzymes with dramatic different and tunable reactivity from catalysts without protein scaffolds.

Dimerized Domain V of Beta2-Glycoprotein I Is Sufficient to Upregulate Procoagulant Activity in PMA-Treated U937 Monocytes and Require Intact Residues in Two Phospholipid-Binding Loops.

Upregulation of the procoagulant activity of monocytes by antibodies to beta2- glycoprotein I (ß2GPI) is one of the mechanisms contributing to thrombosis in antiphospholipid syndrome. Current knowledge about receptors responsible for the upregulation of procoagulant activity by ß2GPI/anti-ß2GPI complexes and their binding sites on ß2GPI is far from complete. We quantified the procoagulant activity expressed by phorbol 12-myristate 13-acetate (PMA)- differentiated U937 cells by measuring clotting kinetics in human plasma exposed to stimulated cells. Cells stimulated with anti-ß2GPI were compared to cells treated with dimerized domain V of ß2GPI (ß2GPI-DV) or point mutants of ß2GPI-DV. We demonstrated that dimerized ß2GPI-DV is sufficient to induce procoagulant activity in monocytes. Using site-directed mutagenesis, we determined that the phospholipid-binding interface on ß2GPI is larger than previously thought and includes Lys308 in ß2GPI-DV. Intact residues in two phospholipid-binding loops of ß2GPI-DV were important for the potentiation of procoagulant activity. We did not detect a correlation between the ability of ß2GPI-DV variants to bind ApoER2 and potentiation of the procoagulant activity of cells. The region on ß2GPI inducing procoagulant activity in monocytes can now be narrowed down to ß2GPI-DV. The ability of ß2GPI-DV dimers to come close to cell membrane and attach to it is important for the stimulation of procoagulant activity.

Covalent Anchor Positions Play an Important Role in Tuning Catalytic Properties of a Rationally Designed MnSalen-containing Metalloenzyme.

Two questions important to the success in metalloenzyme design are how to attach or anchor metal cofactors inside protein scaffolds, and in what way such positioning affects enzymatic properties. We have previously reported a dual anchoring method to position a nonnative cofactor, MnSalen (1), inside the heme cavity of apo sperm whale myoglobin (Mb) and showed that the dual anchoring can increase both the activity and enantioselectivity over the single anchoring methods, making this artificial enzyme an ideal system to address the above questions. Here we report systematic investigations of the effect of different covalent attachment or anchoring positions on reactivity and selectivity of sulfoxidation by the MnSalen-containing Mb enzymes. We have found that changing the left anchor from Y103C to T39C has an almost identical effect of increasing rate by 1.8-fold and increasing selectivity by +14% for S, whether the right anchor is L72C or S108C. At the same time, regardless of the identity of the left anchor, changing the right anchor from S108C to L72C increases rate by 4-fold and selectivity by +66%. The right anchor site was observed to have a greater influence than the left anchor site on the reactivity and selectivity in sulfoxidation of a wide scope of other ortho-, meta- and para- substituted substrates. The 1•Mb(T39C/L72C) showed the highest reactivity (TON up to 2.31 min(-1)) and selectivity (ee% up to 83%) among the different anchoring positions examined. Molecular dynamic simulations indicate that these changes in reactivity and selectivity may be due to the steric effects of the linker arms inside the protein cavity. These results indicate that small differences in the anchor positions can result in significant changes in reactivity and enantioselectivity, probably through steric interactions with substrates when they enter the substrate-binding pocket, and that the effects of right and left anchor positions are independent and additive in nature. The finding that the anchoring arms can influence both the positioning of the cofactor and steric control of substrate entrance will help design better functional metalloenzymes with predicted catalytic activity and selectivity.