Development of Nonheme {FeNO}⁷ Complexes Based on the <i>Pyrococcus furiosus</i> Rubredoxin for Red-Light-Controllable Nitric Oxide Release

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron–sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration

Formation of Nitric Oxide from Nitrite by the Ferriheme <i>b</i> Protein Nitrophorin 7

Formation of Nitric Oxide from Nitrite by the Ferriheme b Protein Nitrophorin

Breaking the Proximal Fe^II–N_His Bond in Heme Proteins through Local Structural Tension: Lessons from the Heme <i>b</i> Proteins Nitrophorin 4, Nitrophorin 7, and Related Site-Directed Mutant Proteins

The factors leading to the breakage of the proximal iron–histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV–vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1–4). Structural considerations led to the generation and spectroscopic investigation of site-directed mutants NP7­(E27V), NP7­(E27Q), NP4­(D70A), and NP2­(V24E). Spectroscopic investigation of these proteins shows that the spatial arrangement of residues Glu27, Phe43, and His60 in the proximal heme pocket of NP7 is the reason for the weakened FeII–His60 bond through steric demand. Spectroscopic investigation of the sample of NP7 reconstituted with 2,4-dimethyldeuterohemin (“symmetric heme”) demonstrated that the heme vinyl substituents are also responsible. Whereas the breaking of the iron–histidine bond is rarely seen among unliganded ferroheme proteins, the breakage of the FeII–His bond upon binding of NO to the sixth coordination site is sometimes observed because of the negative trans effect of NO. However, it is still rare among the heme proteins, which is in contrast to the case for trans liganded nitrosyl model hemes. Thus, the question of which factors determine the FeII–His bond labilization in proteins arises. Surprisingly, mutant NP2­(V24E) turned out to be particularly similar in behavior to sGC; i.e., the FeII–His bond is sensitive to breakage upon NO binding, whereas the unliganded form binds the proximal His at neutral pH. To the best of our knowledge, NP2­(V24E) is the first example in which the ability to use the His-on ↔ His-off switch was engineered into a heme protein by site-directed mutagenesis other than the proximal His itself. Steric tension is, therefore, introduced as a potential structural determinant for proximal FeII–His bond breakage in heme proteins

Formation of the Complex of Nitrite with the Ferriheme <i>b</i> β-Barrel Proteins Nitrophorin 4 and Nitrophorin 7,

The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5, which is an unprecedented case among ferrihemes [He, C., and Knipp, M. (2009) J. Am. Chem. Soc. 131, 12042−12043]. NPs, which comprise a class of unique heme b proteins from the saliva of the blood-sucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result, the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore, X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation of an η1-N nitro complex, which is in contrast to the η1-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15−190 M−1) are comparable to those of metHb and metMb, the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoprotein

On Demand Attachment and Detachment of <i>rac</i>-2-Br-DMNPA Tailoring to Facilitate Chemical Protein Synthesis

Herein, we developed a bifunctional reagent rac-2-Br-DMNPA 2 for the late-stage protection of peptide cysteine. Through the identification of its t-Bu ester 1 as a more competent form under ligation conditions, facile N-terminal and side-chain caging for the model peptide and protein were accomplished. Building upon this, a one-pot ligation and photolysis strategy was applied in the synthesis of the mini-protein chlorotoxin. More importantly, we extended the utility of 2 as a bifunctional linker for traceless solid-phase chemical ligation

Oxidation and Phenolysis of Peptide/Protein C‑Terminal Hydrazides Afford Salicylaldehyde Ester Surrogates for Chemical Protein Synthesis

With the growing popularity of serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL) in chemical protein synthesis, facile and general approaches for the preparation of peptide salicylaldehyde (SAL) esters are urgently needed, especially those viable for obtaining expressed protein SAL esters. Herein, we report the access of SAL ester surrogates from peptide hydrazides (obtained either synthetically or recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoic acid (SAL(−COOH)PDT). The resulting peptide SAL(−COOH)PDT esters can be activated to afford the reactive peptide SAL(−COOH) esters for subsequent STL/CPL. While being operationally simple for both synthetic peptides and expressed proteins, the current strategy facilitates convergent protein synthesis and combined application of STL with NCL. The generality of the strategy is showcased by the N-terminal ubiquitination of the growth arrest and DNA damage-inducible protein (Gadd45a), the efficient synthesis of ubiquitin-like protein 5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesis of a myoglobin (Mb) variant

Repurposing a Nitric Oxide Transport Hemoprotein Nitrophorin 2 for Olefin Cyclopropanation

A growing number of heme proteins have recently been repurposed for catalyzing abiological carbene transfer reactions. Herein, we rationally designed an engineered variant of nitrophorin 2 (NP2)a nitric oxide transport hemoproteinthat catalyzes olefin cyclopropanation with high activity and stereoselectivity. Being a β-barrel protein, the engineered NP2 variant showed a unique substrate preference, in contrast to the mainstream α-helical carbene-transfer heme enzymes like cytochrome P450 enzymes and myoglobin. The catalytic reactions can be carried out on a preparative scale while maintaining the stereoselectivity. The stereoselectivity of the NP2-catalyzed styrene cyclopropanation was further supported by quantum chemical calculations, and the significance of key residues was elucidated. As such, this work establishes NP2 as a robust lipocalin scaffold amenable for carbene-transferase development, complementing the current biocatalytic toolbox

Repurposing a Nitric Oxide Transport Hemoprotein Nitrophorin 2 for Olefin Cyclopropanation

A growing number of heme proteins have recently been repurposed for catalyzing abiological carbene transfer reactions. Herein, we rationally designed an engineered variant of nitrophorin 2 (NP2)a nitric oxide transport hemoproteinthat catalyzes olefin cyclopropanation with high activity and stereoselectivity. Being a β-barrel protein, the engineered NP2 variant showed a unique substrate preference, in contrast to the mainstream α-helical carbene-transfer heme enzymes like cytochrome P450 enzymes and myoglobin. The catalytic reactions can be carried out on a preparative scale while maintaining the stereoselectivity. The stereoselectivity of the NP2-catalyzed styrene cyclopropanation was further supported by quantum chemical calculations, and the significance of key residues was elucidated. As such, this work establishes NP2 as a robust lipocalin scaffold amenable for carbene-transferase development, complementing the current biocatalytic toolbox

Heterogeneous Kinetics of the Carbon Monoxide Association and Dissociation Reaction to Nitrophorin 4 and 7 Coincide with Structural Heterogeneity of the Gate-Loop

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme–CO complexes of NP4, NP4­(D30N), and NP7 as isoelectronic models for the ferriheme–NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-IR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4­(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leu130 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs

Nitrite Dismutase Reaction Mechanism: Kinetic and Spectroscopic Investigation of the Interaction between Nitrophorin and Nitrite

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme <i>b</i> protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium­(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (<i>S</i> = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO₂^– cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO₂^– coordination, with the high-spin configuration (<i>S</i> = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}⁷ intermediate species. The first reaction step, generating the {FeNO}⁷ intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs’ function as nitrite dismutase