Heme protonation affects iron-NO binding in the NO transport protein nitrophorin.

The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm-1, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7.

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

Redox-dependent Ligand Switching in a Sensory Heme-binding GAF Domain of the Cyanobacterium Nostoc sp. PCC7120.

The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The "as isolated" form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin Fe(III) heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of -445 and -453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (Fe(II) state) binds both NO and CO. Cysteine coordination of the as isolated Fe(III) protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF domain. DNA binding of the helix-turn-helix domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.

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 b 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 (S = 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 (S = 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.

A caged substrate peptide for matrix metalloproteinases.

Based on the widely applied fluorogenic peptide FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; Mca = methoxycoumarin-4-acetyl; Dpa = N-3-(2,4-dinitrophenyl)l-α,ß-diaminopropionyl) a caged substrate peptide Ac-Lys-Pro-Leu-Gly-Lys*-Lys-Ala-Arg-NH2 (*, position of the cage group) for matrix metalloproteinases was synthesized and characterized. The synthesis implies the modification of a carbamidated lysine side-chain amine with a photocleavable 2-nitrobenzyl group. Mass spectrometry upon UV irradiation demonstrated the complete photolytic cleavage of the protecting group. Time-resolved laser-flash photolysis at 355 nm in combination with transient absorption spectroscopy determined the biphasic decomposition with τa = 171 ± 3 ms (79%) and τb = 2.9 ± 0.2 ms (21%) at pH 6.0 of the photo induced release of the 2-nitrobenzyl group. The recombinantly expressed catalytic domain of human membrane type I matrix metalloproteinase (MT1-MMP or MMP-14) was used to determine the hydrolysis efficiency of the caged peptide before and after photolysis. It turned out that the cage group sufficiently shields the peptide from peptidase activity, which can be thus controlled by UV light.

THz absorption spectroscopy of solvated ß-lactoglobulin.

The influence of ß-lactoglobulin (ßLG) on the fast sub-picosecond collective hydration dynamics in the solvent was investigated by THz absorption spectroscopy as a function of pH. It is well-known that a change in pH from pH 6 to pH 8 reversibly opens or closes the binding cavity by a transition of the E-F loop. Furthermore, the aggregation of the protein into dimers is affected, which is thought to be triggered by changes in the enzyme's electrostatic potential. Our data reveal that pH has a clear influence on the THz absorption of ßLG. We discuss this influence in light of the changes observed in the sub-psec solute/solvent dynamics when probed by THz spectroscopy, which are, in turn, seen to correlate with changes in the pH value.

Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of membrane type 1 matrix metalloproteinase.

Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space group P4(3)2(1)2, with unit-cell parameters a = 62.99, c = 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (VM) of 2.90 Å(3) Da(-1); the solvent content is estimated to be 57.6%.

Preparation of nitrophorin 7(Δ1-3) from Rhodnius prolixus without start-methionine using recombinant expression in Escherichia coli.

The heterologous recombinant expression of proteins in Escherichia coli without start-methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB-nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl2 gradient.

Expression, purification, and solid-state NMR characterization of the membrane binding heme protein nitrophorin 7 in two electronic spin states.

The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1-3 mutant.

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1-3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

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 victim's 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.

Preparation of cysteine-34-nitroxide spin labeled human α1-microglobulin.

α(1)-Microglobulin (α(1)m) is a protein of yet unresolved function occurring in blood plasma and urine. It consists of a lipocaline type of fold with two cysteine residues forming a disulfide bridge and the third cysteine-34 remaining a free, somewhat reactive thiol. A number of investigations point to an interaction with heme and we have recently reported, that heme binding triggers the formation of a stable α(1)m trimer upon modification of cysteine-34 with 2-iodoacetamide, i.e., [α(1)m(heme)(2)](3) [J.F. Siebel, R.L. Kosinsky, B. Åkerström, M. Knipp, Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α(1)-microglobulin-formation of a [(heme)(2)(α(1)-microglobulin)](3) complex, ChemBioChem 13 (2012) 879-887]. For further structural and functional investigations, an improved purification protocol for α(1)m was sought, in particular yielding an untagged amino acid sequence. The method reported herein improves the speed and the yield of the protein production even when an expression plasmid without tag was applied. Furthermore, for the purpose of future structural studies using electron paramagnetic resonance (EPR) techniques, in accordance to the modification with 2-iodoacetamide (α(1)m(AM)), the protein was modified with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-(2-iodoacetamido)-PROXYL) yielding the nitroxide spin labeled α(1)m(N-O). The extinction coefficient of the protein was calibrated using magnetic circular dichroism (MCD) spectroscopy of tryptophan (ε(280nm)=40,625M(-1)cm(-1)). The parallel quantification by absorbance spectroscopy (protein) and cw-EPR spectroscopy (radical spin) determined the degree of spin labeling to 90%. Characterization of the protein by circular dichroism (CD) spectroscopy and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) upon tryptic digestion further demonstrated the similar fold of α(1)m(AM) and α(1)m(N-O), but also established the modification of cystein-34 as well as the formation of the cysteine-72-cysteine-169 disulfide bond.

Insertion of an H-bonding residue into the distal pocket of the ferriheme protein nitrophorin 4: effect on nitrite-iron 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 H(2)O molecules from the heme cavity prevents the proton donation pathway through Asp30.

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.

Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin α(1)-microglobulin: formation of a [(heme)(2) (α(1)-Microglobulin)](3) complex.

α(1)-Microglobulin (α(1)m) is a 26 kDa plasma and tissue protein belonging to the lipocalin protein family. Previous investigations indicate that the protein interacts with heme and suggest that it has a function in heme metabolism. However, detailed characterizations of the α(1)m-heme interactions are lacking. Here, we report for the first time the preparation and analysis of a stable α(1)m-heme complex upon carbamidomethylation of the reactive Cys34 by using recombinantly expressed human α(1)m. Analytical size-exclusion chromatography coupled with a diode-array absorbance spectrophotometry demonstrates that at first an α(1)m-heme monomer is formed. Subsequently, a second heme triggers oligomerization that leads to trimerization. The resulting (α(1)m[heme](2))(3) complex was characterized by resonance Raman and EPR spectroscopy, which support the presence of two ferrihemes, thus indicating an unusual spin-state admixed ground state with S=(3)/(2), (5)/(2).

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.

Identification of the native N-terminus of the membrane attaching ferriheme protein nitrophorin 7 from Rhodnius prolixus.

All species of the genus Rhodnius have a characteristic red coloration in their salivary glands due to the presence of heme proteins. Some of these secreted proteins, known as nitrophorins (NPs), are responsible for many of the antihemostatic activities of Rhodnius saliva such as anticoagulant and antihistamine. Several NPs have been described (NP1-4 and NP7), where NP7 is the only one with affinity to phospholipid membranes. Computational prediction suggested that NP7 also has an extended N-terminal tail on signal peptide cleavage; however, the complementary DNA does not allow the determination of the exact site of signal peptidase cleavage. On the other hand, according to previous studies, the exact length of the N-terminus has important consequences for the nitric oxide binding properties of NP7. Here, a method was developed to select phospholipid membrane-attaching proteins from homogenized tissue for analysis by mass spectrometry. The method was used to determine the exact N-terminus of the ferriheme protein NP7 from homogenates of the salivary glands of 5th instar nymphal stages of Rhodnius prolixus.

Crystallization and preliminary X-ray crystallographic analysis of the membrane-binding haemprotein nitrophorin 7 from Rhodnius prolixus.

Nitrophorins (nitric oxide transport proteins) are haemproteins originating from the blood-feeding insect Rhodnius prolixus. They consist of an eight-stranded ß-barrel, which classifies them into the lipocalin family. Nitrophorin 7 (NP7) and the E27V mutant protein NP7(E27V) were crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. Data sets for wild-type NP7 and NP7(E27V) were collected to 1.80 Å resolution from single crystals at 100 K using synchrotron radiation. The crystals belonged to space group P2(1), with unit-cell parameters a = 38, b = 67, c = 39 Å, ß = 117°. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient (V(M)) of 2.11 Å(3) Da(-1); the solvent content was estimated to be 41.8%.

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).