Chunmao He

Formation of the Complex of Nitrite with the Ferriheme b 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 eta(1)-N nitro complex, which is in contrast to the eta(1)-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 isoproteins.

Nitrophorins: Nitrite disproportionation reaction and other novel functionalities of insect heme-based nitric oxide transport proteins

Nitrophorins (NPs) comprise a unique class of heme proteins used by the blood‐sucking insect Rhodnius prolixus to deliver the signaling gas molecule NO into the blood vessel of a host during feeding. Upon NO release, histamine can be scavenged by coordination to the heme iron. Although the protein is of similar size as the mammalian globin monomers and shares the same cofactor and proximal histidine coordination, nitrophorin structure, in contrast, is almost entirely composed of a β‐barrel. Comparison of the NO and histamine association constants with the concentrations of both compounds invivo raises concerns about the very simple ligand release model in case of at least some of the NPs. Therefore, novel functionalities of the NPs were sought. As a result, catalysis of the nitrite disproportionation reaction was found, which leads to the formation of NO with nitrite as the sole substrate. This is the first example of a ferriheme protein that can perform this reaction. Furthermore, although NPs stabilize the ferriheme state, a peroxidase reactivity of the cofactor involving the higher oxidation state iron (Compound I/II) was studied with the potential to catalyze the oxidation of histamine and norepinephrine. In contrast to many other heme proteins including the globins, the ferroheme state was found to be extremely sensitive to O2, which is a consequence of the much lower reduction potential of the NPs, so that the 1‐electron reduction of O2 to O  •−2 becomes a thermodynamically favored process. Altogether, the detailed study of the NPs gives insight into the structure‐function relationships required for the targeted delivery of diatomic gas molecules in biology. Moreover, the comparison of the structure‐function relationships of the NPs (NO transporters) with those of the globins (O2 transporters) will help to elucidate the architectural requirement for the respective tasks.

Formation of nitric oxide from nitrite by the ferriheme b protein nitrophorin 7.

Recently, the conversion of nitrite into NO by certain heme proteins, in particular hemoglobin, gained much interest as a physiologically important source of NO in human tissue. However, in an aqueous environment, nitrite reduction at an iron porphyrin occurs either through oxidation of ferroheme to ferriheme or with the assistance of a second substrate molecule. Here we report on the reduction of nitrite in the absence of a second substrate at the heme center of the ferriheme protein nitrophorin 7 (NP7) resulting in the formation of NO and restoration of the ferriheme center. The product was spectroscopically characterized, in particular by resonance Raman and FT-IR spectroscopy. Performing the reaction in the presence of the NO trap 2-(4-trimethylammonio)phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (TMA-PTIO) revealed that continuous NO production is possible, i.e., that NP7 is fully restored upon a single turnover. Thus, NP7 is the first case of a b-type heme that performs reduction of nitrite as a single substrate out of the iron(III) state.

Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction.

The determination of the redox properties of the cofactor in heme proteins provides fundamental insight into the chemical characteristics of this wide-spread class of metalloproteins. For the preparation of the ferroheme state, probably the most widely applied reductant is sodium dithionite, which at neutral pH has a reduction potential well below the reduction potential of most heme centers. In addition to the heme iron, some heme proteins, including the nitrophorins (NPs), contain cysteinecysteine disulfide bonds. In the present study, the effect of dithionite on the disulfides of NP4 and NP7 is addressed. To gain deeper understanding of the disulfide/dithionite reaction, oxidized glutathione (GSSG), as a model system, was incubated with dithionite and the products were characterized by (13)C NMR spectroscopy and reverse phase chromatography in combination with mass spectrometry. This revealed the formation of one equivalent each of thiol (GSH) and glutathione-S-thiosulfate (GSSO(3)(-)). With this background information, the effect of dithionite on the cystines of NP4 and NP7 was studied after trapping of the thiols with para-cloromercurybenzyl sulfonate (p-CMBS) and subsequent matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) where the heterolytic cleavage of the SS bond appears with only 2molar equivalents of the reductant. Furthermore, prolonged electrochemical reduction of NP4 and NP7 in the presence of electrochemical mediators also leads to disulfide breakage. However, due to sterical shielding of the disulfide bridges in NP4 and NP7, the cystine reduction can be largely prevented by the use of stoichiometric amounts of reductant or limited electrochemical reduction. The described disulfide breakage during routine iron reduction is of importance for other heme proteins containing cystine(s).

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.

Complexes of ferriheme nitrophorin 4 with low-molecular weight thiol(ate)s occurring in blood plasma

Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victims tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component L-cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899-905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, L-cysteine, and L-homocysteine and with HS(-) were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by L-cysteine, all four compounds form low-spin Fe(III) complexes with NP4. The weak equilibration constants (167-5200 M(-1)) neither support significant complexation nor the simple displacement of NO in vivo. Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and L-methionine did not form a complex, indicating the specificity of the complexes with L-cysteine and L-homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a ferriheme protein in complex with L-cysteine L-homocysteine.

Insertion of an H‐Bonding Residue into the Distal Pocket of the Ferriheme Protein Nitrophorin 4: Effect on NitriteIron Coordination and Nitrite Disproportionation

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoproteinnitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H‐bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H‐bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible GH loop as a potential H‐bonding residue in the distal heme pocket. Similar to the wild‐type protein, nitrite remains N‐bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand‐rotational orientation exists, which is in fast‐exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H2O molecules from the heme cavity prevents the proton donation pathway through Asp30.

A caged cyanide

A photoactivatable caged cyanide, 1-(2-nitrophenyl)ethyl (NPE) cyanide, was synthesized, which upon irradiation in the near UV releases cyanide. It is demonstrated that the compound can be used to induce formation of the Fe(III)-CN(-) complex in the heme protein nitrophorin 4 from Rhodnius prolixus.