NMR Studies of the Dynamics of High-Spin Nitrophorins: Comparative Studies of NP4 and NP2 at Close to Physiological pH

The β-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect <i>Rhodnius prolixus</i>, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1–NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) and NP2 and NP3 (80% identical). The available crystal structures of NP4 have been used to propose that pH-dependent changes in the conformation of two loops between adjacent β-strands at the front opening of the protein, the A–B and G–H loops, determine the rate of NO release. At pH 7.3, NP4 releases NO 17 times faster than NP2 does. In this work, the aqua complexes of NP4 and NP2 have been investigated by nuclear magnetic resonance (NMR) relaxation measurements to probe the pico- to nanosecond and micro- to millisecond time scale motions at two pH values, 6.5 and 7.3. It is found that NP4-OH₂ is fairly rigid and only residues in the loop regions show dynamics at pH 6.5; at pH 7.3, much more dynamics of the loops and most of the β-strands are observed while the α-helices remain fairly rigid. In comparison, NP2-OH₂ shows much less dynamics, albeit somewhat more than that of the previously reported NP2-NO complex [Muthu, D., Berry, R. E., Zhang, H., and Walker, F. A. (2013) <i>Biochemistry 52</i>, 7910–7925]. The reasons for this major difference between NP4 and NP2 are discussed

NMR Studies of the Dynamics of Nitrophorin 2 Bound to Nitric Oxide

The <i>Rhodnius</i> nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect <i>Rhodnius prolixus</i>, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1–NP4. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A–B and G–H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2–NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2–NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A–B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins

NMR Studies of the Dynamics of High-Spin Nitrophorins: Comparative Studies of NP4 and NP2 at Close to Physiological pH

The β-barrel nitrophorin (NP) heme proteins are found in the saliva of the blood-sucking insect <i>Rhodnius prolixus</i>, which synthesizes and stores nitric oxide (NO) in the salivary glands. NO is bound to iron of the NPs and is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1–NP4, which have sequence similarities in two pairs, NP1 and NP4 (90% identical) and NP2 and NP3 (80% identical). The available crystal structures of NP4 have been used to propose that pH-dependent changes in the conformation of two loops between adjacent β-strands at the front opening of the protein, the A–B and G–H loops, determine the rate of NO release. At pH 7.3, NP4 releases NO 17 times faster than NP2 does. In this work, the aqua complexes of NP4 and NP2 have been investigated by nuclear magnetic resonance (NMR) relaxation measurements to probe the pico- to nanosecond and micro- to millisecond time scale motions at two pH values, 6.5 and 7.3. It is found that NP4-OH₂ is fairly rigid and only residues in the loop regions show dynamics at pH 6.5; at pH 7.3, much more dynamics of the loops and most of the β-strands are observed while the α-helices remain fairly rigid. In comparison, NP2-OH₂ shows much less dynamics, albeit somewhat more than that of the previously reported NP2-NO complex [Muthu, D., Berry, R. E., Zhang, H., and Walker, F. A. (2013) <i>Biochemistry 52</i>, 7910–7925]. The reasons for this major difference between NP4 and NP2 are discussed

Electron Spin Density on the Axial His Ligand of High-Spin and Low-Spin Nitrophorin 2 Probed by Heteronuclear NMR Spectroscopy

The electronic structure of heme proteins is exquisitely tuned by the interaction of the iron center with the axial ligands. NMR studies of paramagnetic heme systems have been focused on the heme signals, but signals from the axial ligands have been rather difficult to detect and assign. We report an extensive assignment of the ¹H, ¹³C and ¹⁵N resonances of the axial His ligand in the NO-carrying protein nitrophorin 2 (NP2) in the paramagnetic high-spin and low-spin forms, as well as in the diamagnetic NO complex. We find that the high-spin protein has σ spin delocalization to all atoms in the axial His57, which decreases in size as the number of bonds between Fe­(III) and the atom in question increases, except that within the His57 imidazole ring the contact shifts are a balance between positive σ and negative π contributions. In contrast, the low-spin protein has π spin delocalization to all atoms of the imidazole ring. Our strategy, adequately combined with a selective residue labeling scheme, represents a straightforward characterization of the electron spin density in heme axial ligands

Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center inside a β-Barrel Protein

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN^–), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect <i>Rhodnius prolixus</i>. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2–NO complex and Type I and II electronic ground states of the NP2–CN^– and NP2–histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN^– and histamine, might be a consequence of the protonation state of the heme carboxyls

Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center inside a β-Barrel Protein

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN^–), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect <i>Rhodnius prolixus</i>. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2–NO complex and Type I and II electronic ground states of the NP2–CN^– and NP2–histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN^– and histamine, might be a consequence of the protonation state of the heme carboxyls