NMR Investigations of Nitrophorin 2 Belt Side Chain Effects on Heme Orientation and Seating of Native N-terminus NP2 and NP2(D1A)

Abstract

Nitrophorin 2, one of the four NO–storing and –releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B), compared to those of NP1 and NP4, which show no preference (A:B ~ 1:1), suggesting that it fits more tightly in the β-barrel protein. In this work we have prepared a series of “Belt” mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole (NMeIm) complexes. The “Belt” mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T and a triple mutant of NP2(D1A), the F42L,L106F,I120T mutant. Although I120 has been mainly considered to be a distal pocket residue, the CδH₃ of I120 lies directly above the heme 3-methyl, at 2.67 Å, of heme orientation B, or 2-vinyl of A, and it thus plays a role as a “Belt” mutant, a role that turns out to be extremely important in creating the strong favoring of the B heme orientation (A:B = 1:14) for NP2(D1A) or 1:12 for (ΔM0)NP2. The results show that the 1D ¹H NMR spectra of the high-spin forms are quite sensitive to changes in the shape of the heme binding cavity. The single mutation I120T eliminates the favorability of the B heme orientation by producing a heme A:B orientation of 1:1, while the single mutation F42A reverses the heme orientation from A:B = 1:14 seen for NP2(D1A) to 10:1 for NP2(D1A,F42A). The most extreme ratio was found for the triple mutant of NP2(D1A), NP2(D1A,F42L,L105F,I120T), in which A:B ~25:1, a ΔG change of about −3.5 kcal/mol or −14.1 kJ/mol with respect to NP2(D1A). The seating of the heme is modified as well in that mutant and several others, by rotations of the heme by up to 4° from the seating observed in NP2(D1A), in order to relieve steric interactions between a vinyl β-carbon and a protein side chain, or to fill a cavity created by replacing a large protein side chain by a much smaller one; the latter was observed for all Tyr→Ala mutants. These relatively small changes in seating have measureable effect on the NMR spectra of the mutants, but are indeed minor in terms of overall seating and reactivity of the NP2(D1A) protein. The ¹H NMR resonances of the hemin substituents of the low-spin NMeIm complexes of NP2(D1A,F42L,L105F,I120T) as well as NP2(D1A,I120T), NP2(D1A,Y104A) and NP2(D1A,F42A) have been assigned using natural abundance ¹H{¹³C} HMQC and ¹H-¹H NOESY spectra.

Introduction

The nitrophorins are a group of nitric oxide (NO) storing and carrying heme proteins found in the saliva of some species of blood-sucking insects [1]. One of these insects is Rhodnius prolixus (the “kissing bug”), the adult insect of which has four salivary nitrophorins (NP1-4 in order of decreasing abundance) that store NO via a ferriheme-nitrosyl complex and keep it stable for long periods of time [2, 3]. The NO is produced by a constituitive nitric oxide synthase (NOS) enzyme which is present in the epithelial cells of the salivary glands [4]. When injected into the tissues of the bug’s victim, a combination of ~100-fold dilution and pH rise (from pH ~5–6 in the salivary glands to pH ~7.3–7.4 in the tissues) cause dissociation of NO. The NO diffuses through the tissues to the nearby capillaries to cause vasodilation (and inhibition of platelet aggregation) to thereby allow more blood to be transported to the site of the wound [5, 6]. In addition, the ferriheme centers of the Rhodnius nitrophorins are able to bind histamine, which is released by mast cells and platelets of the victim in response to the wound. This histamine would otherwise cause swelling, itching, and the beginning of the immune response of the victim; its binding to the nitrophorins hence prevents the insect’s detection for a period of time [7]. These properties of the nitrophorins of the insect assist the bug in obtaining a sufficient blood meal in a minimum amount of time. Of the four Rhodnius nitrophorins of the adult insect, NP2 is unique, for in addition to its NO-releasing and histamine-binding roles, it also has anticoagulant activity, via heme-independent inhibition of the factor Xase complex [8 – 10].

The structures of Rhodnius nitrophorins bound to various ligands have been determined by X-ray crystallography for NP1 [11 – 13], NP2 [14 – 16] and NP4 [17 – 25]. These structures show the heme to be located inside a β-barrel, with the propionate groups protruding into the aqueous medium. This structure is unique for heme proteins, which more commonly have α-helical globin or 4-helix bundle folds [26, 27]. The ferriheme prosthetic group is bound to the protein via a histidine ligand, and the sixth coordination site is available to bind NO, histamine, or other ligands, and if no other ligand is added, water, to yield a high-spin Fe(III) complex. Ligand binding has been investigated by a number of spectroscopic techniques [12, 28 – 46], including infrared [12, 28], resonance Raman [28], EPR [30], NMR [12, 31 – 41] and Mössbauer spectroscopies [41, 43], nuclear inelastic scattering [43], stopped-flow [44] and laser flash [45, 46] kinetics, and spectroelectrochemistry [12, 25, 32, 35, 44]. The nitrophorins store and transport NO via a ferriheme-nitrosyl complex, which is bound to NO with stabilities that facilitate release upon dilution in biological tissues (K_d ~ 5–900 nanomolar) [25]. Typically, other heme proteins have much more positive reduction potentials than the nitrophorins and these ferriheme-nitrosyl complexes are unstable with respect to reduction to Fe(II)-NO in the presence of excess NO [47], which bind NO irreversibly (K_d ~ picomolar – femtomolar [45]).

The nitrophorins all have two conserved leucine residues in the distal pocket where NO, histamine and other ligands can bind, which have their side chains positioned above and in contact with the heme near the iron binding site, as shown in Figure 1, where they may possibly contribute to the extensive distortion of the heme from planarity which is particularly extreme in NP2, but is also present in NP1 and NP4. Unfortunately, it has not been possible to obtain crystal structures of NP3 thus far, but because of the similarity in the protein sequences and the proton NMR shifts of protein side chains of NP2 and NP3 [39], we surmise that NP3 has a similarly nonplanar heme, as well as a heme binding pocket extremely similar to that of NP2. The nonplanarity is believed to affect the Fe^III/Fe^II midpoint potential, and thus the reactivity of the ferriheme center toward NO [32]. The Leu122(123) side chain is situated near the opening to the ligand binding pocket, while the Leu132(133) side chain is buried deep inside the pocket, and is believed to be largely responsible for the heme nonplanarity and midpoint potential [19, 32].

Figure 1.

View of the heme pockets of NP2(D1A)-NH₃PDB file 2EU7 [15] (left), and NP4-NH₃PDB file 1X8P [22] (right), with the residues around the heme, four of which differ between the two proteins, highlighted in CPK mode (Leu106 vs. Phe107 (yellow), Ile120 vs. Thr121 (apple green), Ser40 vs. Ala42 (brown), and Phe42 vs. Leu44 (orange). Also shown are the “Belt” aromatic residues Tyr104(105) (royal blue), Tyr85(Phe87) (pink), Glu53(55) (black), Tyr38(40) (grass green) and the leucines 122(123) (aqua), and 132(133) (olive), all in ball and stick mode.

In addition to the two conserved leucine residues in the NP2 and NP3 distal pocket there is a third residue which also is positioned above and in contact with the heme near the distal ligand binding site, also deep inside the distal pocket. This residue is an isoleucine (I120) for NP2 and NP3 (Figure 1), but a threonine (T121) for NP1 and NP4. The crystal structure of NP2(D1A)-NH₃ (PDB file 2EU7 [15]) shows that the Ile120 Cδ1 methyl carbon is only 3.50 Å from the heme 3-methyl carbon, meaning that their methyl protons are slightly less than 2.59 Å apart, or essentially within van der Waals contact. In contrast, for the B heme orientation of NP4, the Cγ2 methyl carbon of Thr121 is 4.66 Å from the 3-methyl carbon of the B heme orientation, and their protons are 3.32 Å apart (PDB file 1KOI [19]), or 4.36 Å from the 1-methyl carbon of the A heme orientation, and their protons are 3.09 Å apart. This means that Thr121 cannot contribute to the ruffling of the heme of NP4, and indeed, X-ray crystal structures show the heme of NP4 [17–22] is less ruffled than that of NP2 [14, 15, Supporting Information Table S1]. Because of the large difference in size of the side chain of Ile and Thr, as well as the difference in their hydrophobicity, we wondered what the role of this residue is in determining the properties of the nitrophorins, in particular the heme seating and the ratio of heme orientations, A and B, which exist because of the unsymmetrical placement of the two vinyl groups of protohemin IX (Scheme I). In the wild-type nitrophorins, NP1 and NP4 show no preference for A or B (A:B ~1:1), while NP2 has an A:B ratio of 1:25 at equilibrium and NP3 has a ratio of about 1:4. Therefore, we studied the effects of the size of the Ile120/Thr121 residue in detail in a recent publication [38] by preparing the I120T and I120V mutants of wild-type NP2 and the T121S, L and I mutants of wt NP1 and found that the two large side chains produce an equilibrium ratio of A:B isomers which favor the B orientation, 2:3 and 1:2, respectively. However, since the presence of M0 in wt NP2 (hereafter called (M0)NP2) and wt NP1 (hereafter called (M0)NP1) prevents the A-B and G-H loops from closing [35], we have also investigated the I120T mutant of NP2(D1A) and (ΔM0)NP2, as well as a number of other site-directed mutants, in this work.

Scheme I.

Heme orientations A and B, with His57 behind the heme, Ile120 (orange), Leu122 (yellow) and Leu132 (yellow) in front of the heme. --β,δ meso line; — His imidazole plane orientation.

(M0)NP1 and NP4 both have nearly equal ratios of heme orientations A:B, while (M0)NP2 and (M0)NP3 have been reported to have A:B ratios of 1:8 and 1:4 at equilibrium for the high-spin forms, respectively [33]. However, in recent work we have found that with equilibration times of 8 days or more, the A:B ratio of fully-equilibrated (M0)NP2 is actually 1:25 [40], while that of NP2(D1A) is 1:14 [35] and that of native N-terminus NP2 (hereafter called (ΔM0)NP2) is similar, at 1:12 [39]. Thus, for high-resolution, multidimensional NMR studies, NP2 is by far the most favorable Rhodnius NP to investigate, because it has a large difference in the abundance of the two heme isomers [31 – 41]. Thus at pH 7.0 - 7.5 with a ratio of A:B = 1:14, the resonances of the 7.1% A of NP2(D1A) do not interfere with assignment of the protein resonances of the B-orientation heme-containing protein in the multidimensional (2-4D) ¹H,¹³C,¹⁵N NMR experiments, or the use of (ΔM0)NP2 for carrying out dynamics measurements by NMR spectroscopy [48]. For the other three Rhodnius NPs it would be necessary to use the “symmetrical hemin” (2,4-dimethyldeuterohemin) in order to assign the protein resonances. And as will become clear throughout this paper, the two extra carbons of the two vinyl groups actually affect a number of the thermodynamic and spectroscopic properties of the NPs.

In addition to the three distal pocket side chains of all four nitrophorins of the adult insect, discussed above, a number of residues are positioned like a “belt” around the inside of the beta-barrel, as shown in Figure 1, in such a way that they may interact with the heme to affect the heme A:B orientation ratio and the tightness of the heme fit inside the β-barrel, and thus the degree of ruffling of the heme. Among these, there are several sequence differences between NP1, NP4 on one hand and NP2, NP3 on the other, for which the side chains are different in size, for example, A42 as compared to S40, L44 as compared with F42, F107 as compared to L106, and I120 compared to T121, respectively. We have already investigated the S40A mutant of (M0)NP2 and the A42S mutant of (M0)NP1 and found that neither mutation changed the A:B ratio or the seating of the heme, and thus this position is not important in controlling the heme orientation or seating [38]. However, we have also investigated the L44F mutant of (M0)NP1 and find that it has significant chemical exchange processes in the high-spin state that affect the appearance of the 1D NMR spectra of this mutant [36, 38], as well as in the low-spin imidazole complex [38]. In this work we have continued the investigation of the differences in residues between the two groups of nitrophorins, shown in space-filling mode in Figure 1. Many other “belt” residues are conserved, or nearly so, among the nitrophorins, including V24(25), F27(Y28),^* V34(36), E53(55), L55(57), Y85(F86), and Y104(105), and we have probed some of those in this study as well. Finally, the presence of M0 in the case of (M0)NP1, (M0)NP2 and (M0)NP3, as compared to NP4, which has its native N-terminal residue, A1, also provides a major difference in the conformation of the A-B and G-H loops of the four major nitrophorins [35], and possibly also the size and shape of the heme binding pocket. Thus we have investigated this major difference further within the series of proteins presented herein, by preparing a number of “belt” mutants in the D1A construct of NP2, which lacks M0 [35], and in the (ΔM0)NP2 construct, which also lacks M0 and which has its native D1 as first residue [40]. In this work we report the ¹H NMR spectra of the “belt” mutants of high-spin (HS) and low-spin (LS) NP2(D1A) and (ΔM0)NP2.

Materials and Methods

Protein Sample Preparation

Cloning and expression plasmids used for the D1A mutants were those reported previously [14, 35]. Additional mutant genes (for the NP2(D1A,Belt series) where “Belt” = Y38A, F42A, F66A, Y85A, Y104A, I120T, as well as the triple mutant of NP2(D1A), F42L,L106F,I120T, were prepared according to standard genetic engineering methods, and expressed as reported previously [14, 35]. Several additional mutants, where “Belt” = Y104F, Y104H and Y104W, would not fold properly, either in the (M0) or the (D1A) construct, under the many folding conditions tried. Attempts to express and fold NP4(Y105F) also failed. The corresponding (ΔM0)NP2 mutants Y38F, Y85F, Y104A and I120T have been prepared by the methods reported recently [40], in combination with the usual site-directed mutagenesis techniques [14, 35]. The (ΔM0)NP2(Y104F) mutant gene was also constructed and expressed, but the protein could not be folded. The E. coli-expressed mutant proteins were purified as described previously [14, 35, 40], and were stored in lyophilized form, at −80 °C until use. The (ΔM0)NP2 mutant proteins, which were bound to protohemin that was produced during expression, were preliminarily characterized by UV-visible, mass spectrometry (MALDI–TOF) and 1D ¹H NMR spectroscopy. The preliminary 1D NMR spectra showed that all (ΔM0)NP2 mutants contained a small amount of some adventitious ligand that was produced during expression and/or purification, and when these proteins were expressed in small quantity (4–6 L growths), it was found to be impossible to remove this ligand by simple purification techniques used previously [14, 35], which produced variable amounts of low-spin heme protein signals in the high-spin samples.

NMR data collection

NMR samples were prepared in D₂O containing 50 mM phosphate buffer at pH* = 7.0 (pH* indicates that the pH was read using a standard pH meter and was not corrected for the deuterium isotope effect). NMR spectra were collected over the temperature range 25 to 35 °C, and occasionally as high as 37 °C with the proton chemical shifts referenced to residual water, or to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt, DSS [49]. Natural abundance ¹H{¹³C} HMQC experiments were recorded at a proton Larmor frequency of 600.1299902 MHz and 30 °C on a Bruker AVANCE spectrometer, using a 5 mm inverse-detection probe with decoupling during acquisition. A relaxation delay of 0.2 s and refocusing time of 0.0032 s (J = 200 Hz) were used. The WEFT-NOESY experiments, recorded at 600.1299902 MHz and 30 °C on the same spectrometer, utilized a 0.70 s relaxation delay and 0.125 s recovery-delay. The mixing time for the NOESY experiments was 0.012 s. All 2D spectra were collected with 1918 or 3072 data points in t₂ for WEFT-NOESY and HMQC experiments, respectively, and with 512–540 blocks in t₁ with 80–512 scans/block.

Results and Discussion

Use of the “Angle Plots”

Before discussing the ¹H NMR spectra of the mutants of this study we wish to introduce the “angle plots,” which are useful tools for analyzing the NMR spectra of ferriheme proteins having at least one histidine or exogenous imidazole ligand bound to iron(III). The “angle plots” are based on the effect of spin delocalization from unpaired electrons in the d_xz and/or d_yz (d_π) orbitals of paramagnetic Fe(III) to the porphyrin ring by L→Fe π donation, in combination with P→Fe π donation to the same d_π orbital, where L represents the histidine imidazole nitrogen π orbital nodal plane, and P represents the porphyrin 3e(π) orbital of proper symmetry to interact with both the particular d_π orbital and the L π orbital [31, 50]. These “angle plots” were developed first for LS ferriheme proteins [50], and were later extended to HS ferrihemes [31]. (They have also been developed for methionine-ligated ferrihemes [51, 52], since the orientation of the π-symmetry p-orbital nodal plane of the sulfur plays the same role for cytochromes c as does the π-symmetry p-orbital nodal plane of the binding nitrogen of the imidazole ring of histidine ligands. When both His and Met are present, it is the Met π-symmetry nodal plane orbital which dominates the pattern of spin delocalization.) The idea behind the “angle plots” is that one can write two possible limiting structures of the 3e(π) molecular orbitals of the porphyrin ring, one set of which has a nodal plane passing through opposite nitrogen atoms, and the other set of which has a nodal plane passing through opposite meso-carbons, and thus oriented at a 45° angle to the first mentioned set [50 – 54]. From those two, one can realize that it should also be possible for the nodal plane to take on the orientation of the other two porphyrin nitrogens, or the other two meso positions, thus covering a total of 180°, and in fact, any arbitrary orientation within that 180°. From that point it is only a small leap of understanding to realize that a p-orbital on a ligand nitrogen which is part of an aromatic ring (like that of the nitrogen of the imidazole ring of histidine which binds to Fe) can control the delocalization of spin density from the d_xz or d_yz orbital of Fe(III) to specific points on the periphery of the porphyrin ring that will thus have more or less spin density delocalized to each position, depending on the particular angle of that nodal plane. Thus, our original hypothesis can be said to be that the nodal plane of a π-donor axial ligand binding to a heme defines the orientation of the e-symmetry d_π orbital of Fe and the nodal plane of the 3e(π) orbital of the porphyrin ring which determine the pattern of spin density delocalization to heme substituents. For low-spin ferrihemes, the electron spin of the half-filled d_π orbital can thus be delocalized efficiently to the porphyrin ring [50], while for high-spin ferrihemes, with 5 unpaired electrons, one of the d_π orbitals will interact with the axial ligand, while the other interacts with the porphyrin ring, thus introducing a 90° shift into the “angle plot” of HS as compared to LS ferrihemes [31, 55]. The “angle plots” are useful for answering questions about heme seating, since they relate the order and relative spacings of the methyl resonances of protohemin-containing proteins of either S = 5/2 (Figure 2), or S = 1/2 spin state (Supporting Information Figure S1), to the angle of the imidazole nodal plane of the coordinated histidine [55] (or a planar exogenous ligand used to produce a low-spin complex).

Figure 2.

“Angle Plot” for high-spin ferrihemes [31, 55], adjusted to the chemical shift scale of the high-spin NP2(D1A) complex, which has 5M and 8M at the same chemical shift. This is the best average angle plot that can be constructed for the NP2(D1A) mutants of this study, although the chemical shift scale varies somewhat for each mutant. The polarity of the heme pocket differs for each mutant, likely because of different numbers of water molecules being present in place of the protein side chain originally present in NP2(D1A) or (ΔM0)NP2. Lines 1, 3, 5 and 8 represent the chemical shifts of the four methyl groups of protohemin, as a function of the angle of the axial His imidazole plane from the x-axis of the heme in A or B orientation shown in Scheme 1, projected on the heme, as viewed from above. The angles of the axial His imidazole plane shown in Scheme 1 are projected onto the angle plot at 125° and 145°, and a third line is provided at 135°, the crystallographic angle for NP2(D1A), PDB file 2EU7 [15]. The average of the four lines is also plotted (dashed line).

A remarkable range of ferriheme proteins has been found to obey the “angle plots” [50 – 59]. In these angle plots, we have not made corrections for the presence of aromatic residues near the heme methyls, because for mainly α-helical heme proteins (myoglobins, cytochromes b₅) there are very few aromatic residues near the hemes, and all of those which are present are phenylalanines; when present, they tend to be at oblique angles to the heme methyls, and thus are not properly positioned to contribute to a ring current shift (see below). Peroxidases and cytochromes c, both of which tend to have a number of tyrosines and tryptophans, as well as phenylalanines, but for peroxidases only two Phe (and no Tyr or Trp) are positioned so as to cause weak ring current shifts, for 5M and 8M (F172 and 179, respectively for horseradish peroxidase, PDB file 1ATJ), while the two near 1M (F142, F143) are too far away to have much effect, and the two near 3M (F45, F152) are at quite oblique angles. For cytochromes c Trp59 and Tyr 67 are near 8M, Tyr 46 is near 5M and Phe10 is near 1M (tuna, PDB file 3CYT), and for horse heart cyt c (PDB file 1HRC) it is the same, except that Phe46 is at a very oblique angle and probably does not contribute significantly. In contrast, for the β-barrel-structured nitrophorins, there is one or more aromatic residue near each of the heme methyls (F27 near 1M, F42 near 3M, Y85, Y104, and F66 near 5M, and Y38 and F66 near 8M (Figure 1), and similarly for NP1 and NP4, except for changes in the residue numbers and F27 and Y85 of NP2 becoming Y28 and F86 for NP1 and NP4), for both heme orientation B and, with slight rearrangements of the angles of F27 and F42, for heme orientation A.

In a paper which also develops DFT methods for methane molecules placed in a variety of positions near aromatic rings of the type found in proteins and nucleic acids, Case [60], and much earlier Haigh and Maillon [61] have reviewed the empirical equations developed from Hückel molecular orbital theory for calculating ring currents near specific sites in proteins. We have used the Hückel approach to evaluate the ring current shifts resulting from the aromatic residues near each of the four heme methyls of NP2 in the B heme orientation. The general form expected for ring current contributions to chemical shifts from the Hückel point of view is

$${\mathrm{\sigma }}_{\text{rc}}=\mathbf{i}\text{BG}(\mathrm{r})$$ (1)

where r is the vector from the observed proton to the aromatic ring, G(r) is a geometric factor and i and B are constants. The geometric factor is

$$\mathrm{G}(\mathrm{r})=\sum _{ij}{s}_{ij}\{1/r_i^3+1/r_j^3\}$$ (2)

where r_i and r_j are the distances from ring atoms i and j to the proton and s_ij is the area of the triangle formed by atoms i and j and the proton point projected onto the plane of the aromatic ring. The sum is over the bonds in the ring. For a methyl group, which spins very rapidly at solution NMR temperatures, all three protons must be treated.

Groups which are nearly perpendicular to the proton or group of protons do not contribute, although they would contribute to deshielding in the Johnson-Bovey treatment of ring currents [62] found in many organic textbooks. Thus, in the case of NP2, Y104 and F66 would not be included because the protons of interest (heme methyl protons) lie far from the perimeter of the ring of the aromatic residue, and are perpendicular to the heme methyl. Thus, we have calculated the values of ζ_rc for F27 near 1M, F42 near 3M, Y85 near 5M and Y38 near 8M, and find that ζ_rc for 1M is −0.66 ppm, for 3M is −0.54 ppm, for 5M is −0.52, and for 8M is −0.46, or as a summary, the ring current contributions to the nitrophorins are ζ_rc ~ −0.5 ± 0.1 ppm for all four methyls, which is certainly within any experimental error that we might realistically assign to the chemical shifts obtained from the angle plot. Therefore, for the NPs, although there appear to be ring currents, they act as a constant contribution that simply shifts the numerical ppm scale. Thus, contributions of aromatic ring currents from phenylalanines, tyrosines and tryptophans are not considered in the angle plots. It has never been claimed that the angle plots predict the chemical shifts to high accuracy; that is the reason that the chemical shift scale was removed soon after publication of the original paper [50, 55]. However, with regard to the angles where heme methyl resonances cross each other, we should expect that in the vast majority of cases, those crossing points should be useful indicators of whether the seating of the heme remains the same, or changes with a particular mutation, because they arise from the angular relationship of the heme methyls to each other and the orientation to the nodal plane of the ligand.

If one of those aromatic residues of NP2(D1A) or (ΔM0)NP2 is mutated to Ala, two things will happen simultaneously: The ring current shift of the aromatic residue will be removed, and a number of water molecules will undoubtedly enter to fill the hole created by removing the large side chain. Those water molecules are expected to change the polarity of the region where they are inserted.

In the case of high-spin ferriheme proteins, since the histidine ligand is provided by the protein, it likely is not able to change its orientation significantly and still be able to bind to the metal, because it is locked into a particular rotamer and cannot rotate significantly about its Nε-Fe bond. In these cases, it will likely be the heme ring which rotates, thus changing its “seating” in order to fit into the mutated heme pocket shape, rather than the histidine-imidazole. Some rotations may be by a relatively small number of degrees, as is the usual case for the nitrophorins (as marked on the two angle plots), while others can be very large (~110°), as for one of the heme seatings of Pseudomonis aeruginosa heme oxygenase bound to its substrate (heme) [56].

For the nitrophorins, X-ray crystallography has shown that His57 (His59 for NP1 and NP4) is oriented so that its imidazole plane lies within ±1° of the β,δ-meso line [11–23] or 135±1° from the x-axis of Scheme I, measured in a counterclockwise direction for heme orientation A, and a clockwise direction for B. Note that φ = 135° is the precise angle where the chemical shifts of 8M and 5M are calculated to cross each other (Figure 2 for high-spin, Supporting Information Figure S1 for low-spin). Thus if 8M and 5M of a high-spin ferriheme complex have the same chemical shift, we know that the angle is precisely 135°. For example, for NP2(D1A) 5M and 8M occur together at 64.3 ppm. X-ray crystallography of NP2(D1A)-NH₃ (a low-spin complex, but it still tells us the orientation of His57) shows that the His57 ligand plane lies exactly over the meso-β – meso-δ line, in the LS case for the exogenous L = NH₃ or CN⁻which contribute no ligand plane to the complex. For L = NMeIm, however, we have found during our studies of the nitrophorins that this exogenous ligand (or any other exogenous imidazole, including ImH, 5MeImH and 2MeImH), rather than His57(59) or an average of the two, dominates the NMR chemical shifts of the heme methyls, and NMeIm usually sits at φ ~150–165° for B, or 105–120° for A, depending on the mutation. For the “parent,” NP2(D1A)-NMeIm or (ΔM0)NP2-NMeIm, the angle is 161–162° for B, 109–110° for A, based on the relative spacings worked out for the quantitative scale for B, Figure 2. The predicted shifts for A are quite similar to the observed shifts, though not identical. This is, in the opinion of the authors, why it is not worthwhile trying to construct exact scales for a given protein, as we have in this particular case at the insistence of a reviewer, because they will never fit another protein exactly. While the low-spin complexes do not confirm for us what the orientation of the His57 imidazole plane is, they provide unambiguous assignments of most or all of the heme substituents, which can readily be transferred back to the high-spin complexes, as discussed below.

If the chemical shifts of 8M and 5M are not the same, then the heme methyl resonances can be assigned by “walking around the heme” of the low-spin complexes, using NOESY crosspeaks between adjacent heme substituents, and natural abundance ¹H{¹³C} HMQC spectra, which clearly identify heme methyls [63] (as well as propionate Hα and Hβ, but less so vinyl Hα and Hβ, which are often not well-resolved). These HMQC assignments are particularly important for the substituents which are buried in the diamagnetic protein envelope, and it is easy to tell a methyl from a methylene. This can only be done for the low-spin complexes because of the rapid electron spin relaxation of the high-spin complexes, and thus it is typically necessary to add NMeIm, or some other not-too-strongly-binding ligand, so that the low-spin methyl assignments can be made and then can be transferred to the high-spin spectra by 1D saturation transfer difference (STD) spectroscopy. (The STD experiments require a separate sample, which has ¼ or less of an equivalent of NMeIm present, so as to be able to observe both HS (broad) and LS (much sharper) resonances.) The assignments can then be used to determine the angle of the His imidazole nodal plane with respect to the heme axis system. Since the changes in angle are relatively small (1–4°), for HS samples (Figure 2) heme orientational isomer A always has a larger methyl resonance spread than B when both heme orientations are present to an abundance where the methyls of both can be assigned. The A heme orientation usually has order 5>8>1>3 or 5=8>1>3, while B has order 8>5>1>3 or 8=5>1>3. (Note that these orders are specifically for the nitrophorins, and may be entirely different for other heme proteins!)

It should be noted that the “angle plots” for 5M and 1M for both HS and LS ferrihemes have the same phase (they are 180° apart on the heme ring), yet the plot for 1M is offset, thus always predicting that 1M will have a smaller chemical shift than 5M. Less obvious, because of the difference in phase of the 3M and 8M lines, is the fact that the 3M plot is also offset to lower chemical shift from the 8M plot. These offsets were introduced when the “angle plots” were created, because of the substituent effects of the vinyls, as compared to the alkyl side chains of the propionates [55], since protohemin bis-cyanide has methyl chemical shifts in the order 8~5>3≳1 (16.3, 15.9, 12.3, 10.3 ppm) [64]; thus offsets of some amount between 5M and 1M, and the same amount (although it does not have to be the same) between 8M and 3M at 0° and 180° were introduced into the “angle plots” [50]. Potential changes in this separation, especially for 5M and 1M were discussed (especially in regard to cytochrome c hemes, where the vinyls have become ethyl groups, with the substituent effect having decreased to nearly zero), and shown in the first “angle plot” paper [55]. The numerical chemical shift scale was later removed to increase generality [55], but we have continued to think of 5M and 1M as having a significant difference in chemical shift at any angle of the axial ligand plane. At times we have found that this was experimentally not the case, even with 1M > 5M for the B heme orientation of a few specific mutants [38]. For both HS and LS mutants presented in this paper, it is clear that the 5M – 1M chemical shift difference actually varies by quite a bit, as seen below in the tables, 9.2–1.7 ppm for HS NPs, 2.3 – 0.97 for LS NPs. This variation may be indicative of changes in the degree of ruffling of the heme, or to the degree of conjugation of the vinyl groups with the heme, or to water molecules changing the polarity near one methyl and not another, or to other factors which have not as yet been identified. Further study of additional mutants will be required in order to understand the reason(s) for the variation. Until that time we will continue to use the “angle plots” shown in Figure 2 or Supporting Information Figures S1.

NMR Characterization of the High-Spin Complexes of the NP2(D1A,Belt) and (ΔM0)NP2(Belt) Mutants

We have shown, by preparation of the D1A mutant of NP2, for which the E. coli methionine aminopeptidase is able to cleave the Met0 [35] as it does for NP4 (whose native first residue is alanine), that the large Met0 side chain interferes with closure of the A-B loop. This A-B loop, in the absence of Met0, has been shown to take up additional conformations [48], which, along with the G-H loop [17–22], have been suggested to reduce the rates of NO and histamine release [44, 45] and of heme A:B orientational equilibration at low pH [35]. The D1A mutant of NP2 has a significant effect on heme orientation (A:B ~1:14 [35] as compared to 1:25 for (M0)NP2 [40]) and axial ligand complex stability [35, 40], but a very small effect on heme seating [39], as reported previously [35]. More recently we prepared native N-terminus NP2 (hereafter called (ΔM0)NP2), and find that its A:B heme orientation ratio is similar, 1:12 [40]. One of our first findings about the two N-terminus proteins was that the NP2(D1A) and (ΔM0)NP2 1D ¹H NMR spectra are essentially identical in the high-spin (S = 5/2) (Figure 3) and also the low-spin (S = 1/2) states [40], thus making it possible to compare NMR spectra of mutants of NP2 in the two constructs directly.

Figure 3.

¹H NMR spectra of high-spin NP2(D1A), (ΔM0)NP2 and the NP2(D1A,Belt) where “Belt” represents F42A, Y104A, Y85A, Y38A, or (ΔM0)NP2(Belt) where “Belt” represents Y104A, Y85F, Y38F high-spin complexes, as well as distal pocket mutant I120T with both N-termini (A1 and D1), and proximal pocket mutant F66A with the D1A N-terminus, all recorded at 600 MHz, pH* 7.0 and 25 °C, showing large effects resulting from mutation of Y38, Y104 or Y85 to A (but much smaller changes, and somewhat smaller change in heme seating upon mutation of F66 to A, but very large change in heme orientation ratio on mutation of I120 to T (A:B ~1:1), of F42 to A, (A:B ~10:1), of Y104 to A (A:B ~1:10) and of Y85 to A (A:B ~14:1). For both the I120T and F42A mutants the spectra are sharp, suggesting that there is little or no dynamics involved in heme seating. For the Y104A and Y85A mutants part of the propionate resonances are very broad, suggesting that rotations about the β-pyrrole-αCH₂ bonds are in the intermediate exchange regime. As can be seen, most (ΔM0)NP2 samples showed the presence of low-spin impurities that could not be removed from these small samples. The A and B heme orientations appear to have rather different His57 orientations in some mutants (I120T, F42A, Y104A, Y85A), as is seen not only by the difference in chemical shift between the resonances of methyls 5 and 8 for A and B, but also by the difference in His β-CH chemical shift (18 to 22 ppm) for B vs A, respectively. These differences suggest that the heme seating is somewhat different for A and B. All samples were maintained at room temperature for at least 8 days before recording the spectra to insure that the equilibrium A:B ratio had been reached, and in some cases for considerably longer periods of time; the parent of this series, NP2(D1A) required equilibration for over three months to achieve that goal.

Although most of the mutants prepared in this work in the NP2(D1A) and (ΔM0)NP2 series involve residues whose side chains interact directly with the edges of the heme (“Belt” mutants), the NP2(D1A,I120T), (ΔM0)NP2(I120T) and NP2(D1A,F66A) mutant proteins were also prepared. It can be seen in Figure 3 that for the I120T mutants of each N-terminus, the 1D ¹H NMR spectra are also almost identical (except for the small impurity peaks of the (ΔM0) mutants), and these two mutants each have approximate 1:1 A:B heme orientation ratios, as compared to the 2:3 A:B heme ratio of the same mutant of the M0 series [38]. However, assignment of the heme resonances of NP2(D1A,I120T) by saturation transfer difference spectroscopy (Supporting Information Figure S2) showed that the heme seatings of A and B are actually fairly similar to those seen in previous mutants of the M0 series [38]: the 5,8M of A are fairly close together (0.8 ppm), suggesting a His57 ligand plane orientation of about 133°, while the 8,5M of B are even closer together, only 0.3 ppm apart, 8M of B at 59.5 and 5M of B at 59.2 ppm at 30 °C, suggesting a His57 ligand plane orientation of a little more than 135° for B. We expect that these His57 imidazole plane orientation changes for A and B represent changes in heme seating, rather than changes in the angles of the side chain rotamer of His57, and thus in this case a clockwise rotation of the heme of about 2° for A, and a counterclockwise rotation of the heme by less than 1° for B, as we look down at the heme from the distal pocket. Such a rotation, with the large I120 side chain gone, would relieve the close distance of 2.68 Å between the 4-vinyl-β-cis proton and the Leu55 Cδ2 methyl protons of the B heme orientation, and the much more important 2-vinyl-β-trans interaction with the phenyl ring of Phe42 of the A orientation. Both the difference in A:B ratio and the seating differences for the A and B isomers are presumably a result of the A-B and G-H loops being able to close in the NP2(D1A) and (ΔM0)NP2 series, thus decreasing the size of the distal pocket in comparison to that of (M0)NP2 [38]. The His57 β-CH resonances at near 18 and 20 ppm for B and A, respectively, clearly confirm that the A:B ratio is close to 1:1. Although the two resonances have different heights and linewidths, their integrated ratios are essentially identical.

The following NP2(D1A) mutants were prepared to probe the importance of the “Belt” of aromatic residues around the heme: NP2(D1A,Y38A), NP2(D1A,F42A), NP2(D1A,Y85A), NP2(D1A,Y104A) and, although not part of the “Belt”, NP2(D1A,F66A), where F66 is beneath the heme, with the phenyl ring in a nearly parallel plane to that of the imidazole ring of His57, the protein-provided heme ligand. For (ΔM0)NP2 the following “Belt” mutants were prepared for comparison to the NP2(D1A) “Belt” mutants, some to probe the importance of the tyrosyl as compared to the phenyl side chain: (ΔM0)NP2(Y38F), (ΔM0)NP2(Y85F) (NP1 and NP4 have F at this position), and (ΔM0)NP2(Y104A). As mentioned in the Materials and Methods section, although both the (ΔM0)NP2(Y104F) and NP4(Y105F) genes were created and expressed, no correctly-folded protein could be obtained in either case. This was also found to be the case for (M0)NP2(Y104F) and NP2(D1A,Y104F) [A. M. Goren, unpublished results]. Thus, loss of the hydrogen-bond between Y104(105) and E53(55) carboxyl, shown in an earlier publication [37] and reprinted in Supporting Information Figure S4, has a significant destabilizing effect on the protein. However, replacement of Y104(105) with the much smaller Ala did produce correctly folded proteins. The fact that these Y→A mutant proteins folded and incorporated heme suggests that the large cavity created by removal of the tyrosine side chain allows space for multiple water molecules to enter the calyx and stabilize the charge on the carboxylate of Glu53 at pH values above its pK_a (~5.8 [25]). The chemical shifts of the high-spin NP2 mutant heme protons of the methyls, vinyls and H57 β-CH, the order and spread of the heme methyl resonances, and the seating change, if any, are summarized in Table 1.

Table 1.

¹H chemical shifts of the heme resonances of the high-spin complexes of NP2(D1A) and (ΔM0)NP2 mutants at 25 °C and pH 7.0.

Protein, Isomer	NP2(D1A) B	(ΔM0) NP2 B	NP2 (D1A, I120T) A	NP2 (D1A, I120T) B	(ΔM0) NP2 (I120T) A	(ΔM0) NP2 (I120T) B	NP2 (D1A, F42A) A	NP2 (D1A, F66A) B	(ΔM0) NP2 (Y85F) B
A:B ratio	1:14	1:12	1:1	1:1	1:1	1:1	10:1	1:20	1:12
1M, ppm	57.9	57.9	62.0	55.5	62.0	54.1	58.2	58.8	60.3
3M, ppm	48.2	48.4	50.2	52.0	49.8	50.4	52.5	49.5	50.4
5M, ppm	64.3	64.5	65.8	59.2	66.0	58.4	67.4	66.9	65.7
8M, ppm	64.3	64.5	65.0	59.5	65.0	61.0	67.4	68.7	65.7
2Hα, ppm	44.8	45.2	49.5	41.3	48.5	41.5	45.3	40.7	41.9
4Hα, ppm	33.1	33.6	40.4	38.8	40.5	38.5	47.1	34.2	34.8
H57 β-CH, ppm	18.6	18.4	19.5	17.2	20.0	17.6	22.0	18.3	18.4
Order	8=5>1>3	8=5>1>3	5>8>1>3	8>5>1>3	5>8>1>3	8>5>1>3	5=8>1>3	8>5>1>3	8=5>1>3
Av. Shift, ppm	58.7	58.8	60.8	56.6	60.7	56.0	61.4	61.0	60.5
Spread, ppm	16.1	16.1	15.6	7.5	11.2	10.6	14.9	19.2	15.3
δ5M – δ1M, ppm	6.4	6.6	3.8	3.7	4.0	4.3	9.2	8.1	5.4
H57 imidazole Plane Angle	135°	135°	<135°	>135°	<135°	>135°	135°	>135°	135°
Heme seating change	No Change	No change	Rotated cwa ~1°	Rotated ccwa ~1°	Rotated cwa ~2°	Rotated ccwa ~1°	No change	Rotated ccwa 3–4°	No change

Protein, Isomer	(ΔM0)NP2 (Y38F) B	NP2(D1A, Y38A) A	NP2(D1A, Y38A) B	NP2(D1A, Y104A) B	(ΔM0)NP2 (Y104A) B	NP2(D1A, Y85A) B	NP2(D1A,F42L, L106F,I120T) A
A:B ratio	~1:3	~1:1	~1:1	~1:10	~1:12	1:14	~25:1
1M, ppm	57.8	58.8	58.8	63.3	63.4	63.0	60.5
3M, ppm	48.1	53.7	56.1	51.5	52.0	50.8	52.3
5M, ppm	64.3	64.9	60.5	71.5	72.2	72.0	66.0
8M, ppm	64.3	62.5	62.5	68.5	69.3	68.8	65.2
2Hα, ppm	38.8	−	−	38.2	38.2	38.0	55.2
4Hα, ppm	33.2	−	−	55.2	55.2	~51.7	42.5
H57 β-CH	17.9	19.7	19.7	21.2	21.2	23.0	20.3
Order	8=5>1>3	5>8>1>3	8>5>1>3	5>8>1>3	5>8>1>3	5>8>1>3	5>8>1>3
Av. Shift, ppm	58.5	57.5	59.5	63.7	64.2	63.7	61.0
Spread, ppm	16.2	11.2	6.4	20.0	20.2	21.2	13.7
δ5M – δ1M, ppm	6.5	6.1	1.7	8.2	8.8	9.0	5.5
H57 imidazole Plane Angle	135°	132°	138–139°	133°	133°	132°	134°
Heme seating change	No change	Rotated cwa ~3°	Rotated ccwa ~3–4°	Rotated cwa ~2°	Rotated cwa ~2°	Rotated cwa ~3°	Rotated cwa ~1°

^a)cw/ccw = clockwise/counterclockwise rotation as seen from above the heme (distal pocket side of the heme where L122, I120 and L132 lie in Scheme I).

For the NP2(D1A,F66A) mutant, the A:B ratio is similar to that of the parent, of the order of A:B = 1:14, and there is only a small angular change in the heme seating of about 2°. The His57 β-CH resonance is at slightly below 20 ppm, also consistent with the B heme orientation. In comparison, although the spectra have some similarities with regard to the pattern of heme methyl resonances, the ones in which “Belt” is Y104A and Y85A were quite difficult to assign to A or B. The larger chemical shift ranges of the NP2(D1A,Y104A), (ΔM0)NP2(Y104A) and NP2(D1A,Y85A) mutants suggested to us early in our study that the major heme orientation isomer was A, as did the H57 β-CH chemical shift of 21 ppm or greater, while the small peak at about 75 ppm of each spectrum suggested that it represented the 5,8-methyl resonances of the minor A heme orientation, while the major isomer was B. In addition, the larger chemical shift range of 21 ppm or larger for the heme methyl resonances could be simply a result of the removal of the tyrosine –OH. We had seen decreases in chemical shift ranges among a number of mutants where a Thr –OH was substituted for a methyl group of Leu or Val, and the reverse where a Ser –OH was replaced by a methyl of Ala [38]. However, in the Tyr to Ala mutants of this study, the change is in the reverse direction. But it is likely that the removal of the large protein side chain leaves extra space which could be filled by water molecules, thus possibly reversing the trend seen previously [38]. Because of the ambiguity in heme orientation, 1D saturation transfer difference (STD) experiments were conducted to assign the heme methyls of NP2(D1A,Y104A). It was found, as shown in Supporting Information Figure S6, that the high-spin heme methyl order is 5>8>1>3, which has, throughout this study, been diagnostic of the A heme orientation. However, assignment of the natural abundance ¹H{¹³C} HMQC and NOESY spectra of the low-spin NMeIm complex of NP2(D1A,Y104A), shown in Supporting Information Figure S5, was only consistent with it being the B isomer, since there are clear NOEs from I120 CδH₃ to 3M and 2Hα, and from L132 CδH₃ to 2Hα, which are only possible for the B heme orientation. Thus the STD data alone might have convinced us that the major orientation is A, but the NOEs for the LS NMeIm complex confirm that the major orientation is actually B, and thus the 5>8>1>3 order is a special case for this mutant and the Y85A mutant. Because of the similarity in the chemical shifts of (ΔM0)NP2(Y104A) to those of NP2(D1A,Y104A) we can be sure that B is the major heme orientation for that mutant as well. And because of the very similar spectral pattern for NP2(D1A,Y85A) it is reasonable to assume that this mutant also has B as the major heme orientation, although we have not investigated it in as much detail.

An interesting finding for the high-spin Y104A mutants is that the 4Hα chemical shift is 55.2 ppm, as shown in Figure 4, which summarizes the information obtained from the STD data of Supporting Figure S5. 55.2 ppm is the highest value of the chemical shift for 4Hα observed thus far for the nitrophorins. It is higher, by far, than the chemical shift of 2Hα, 38.2 ppm, and thus tells us that there is significantly more spin density at the 4-α-vinyl carbon. We see this effect for the Y104A and Y85A mutants, and for the triple mutant of NP2(D1A) discussed below, but in that case, for the 2-vinyl group. These mutants thus give us an idea as to the importance of vinyl in- or out-of-plane position with respect to the heme ring.

Figure 4.

1D ¹H NMR spectrum of NP2(D1A,Y104A) at 25 °C, showing the assignments of resonances from Supporting Information Figure S6. By process of elimination, the 7Hα CH₂ resonances must be those at 49.5 and 25.5 ppm. Note that at 25 °C the existence of two broad peaks at 50.5 and 49.5 ppm and two somewhat sharper peaks at 27 and 25.5 ppm is much more evident than in the saturation transfer/NOE difference spectra of Figure S5, which were recorded at 30 °C. The closeness of each of these two 7Hα peaks to those of 6Hα probably means that both resonances were irradiated and showed difference spectra in Figure S6. Small resonances at 74.8, 62.8 ppm, and possibly at 41.7 and 22.1 ppm, are from the minor A heme orientation.

For the major B heme orientations of NP2(D1A,Y104A) and NP2(D1A,Y85A), the heme seating is changed so that there is a 3–4 ppm difference in chemical shift for 5M and 8M, probably indicating a change in the heme seating of about 2° for the major B orientation of the Y104A mutants, and 3° for the Y85A mutant, in both cases by rotation of the heme in a clockwise direction, rather than the usually-observed counterclockwise direction for the B heme orientation. Removal of the large phenol side chain of Tyr104 probably allows Glu53 to change its position inside the calyx (see Supporting Information Figure S4), thus creating additional space for the 4-vinyl group that causes the small changes in heme seating observed for these mutants, yet leaves the A:B heme orientation unchanged from that of the NP2(D1A) or (ΔM0)NP2 parent. Thus, replacing the large phenol side chain of tyrosine with the methyl group of alanine creates additional space within the calyx which retains the favored B heme orientation, albeit with a slightly modified heme seating. However, since Y104(105) and Y85(F86) are common to all four adult Rhodnius nitrophorins, the properties of these mutants tell us nothing about the relative heme orientation stabilities of the NP1/NP4 vs. NP2/NP3 pairs.

For “Belt” = Y38F, the heme seating is similar to that of the (ΔM0)NP2 parent, as shown in Figure 3 for (ΔM0)NP2 and (ΔM0)NP2(Y38F). In an earlier study of the peroxidase activity of (M0)NP2, (M0)NP2(Y38F) was prepared, and when treated with hydrogen peroxide or peroxyacetic acid, gave rise to a different tyrosyl radical decomposition product than did (M0)NP2 itself [65]. This radical was shown to be located on Y85, by the fact that essentially no radical product was formed when the (M0)NP2(Y38F,Y85F) double mutant was reacted with hydrogen peroxide or peroxyacetic acid [65].

For “Belt” = Y38A, the heme seating is changed significantly, with both A and B having different seatings than expected for a 135° angle for the H57 imidazole plane orientation, angles that are smaller than 135° for A and larger than 135° for B. In addition, the A:B ratio is close to 1:1, and there is little or no dynamics observed. At well over 4 Å for the four closest carbons of the phenol ring of Y38 to the heme, it is too far below the heme to form a π complex in the region of pyrrole ring IV, with 8M and 7P being the nearest substituents. The major difference in the NMR spectra of NP2(D1A,Y38A) and (ΔM0)NP2(Y38F) suggests that for the Y38A mutant, the β-barrel is able to collapse toward the heme to take up much of the space lost by removal of the Tyr side chain, thus changing the NMR spectrum markedly. In any case, this is a second case, after the I120T mutant, where the extra stability of the B heme orientation of NP2 is lost. However, since Y38(40) is common to all four nitrophorins of the adult insect, this mutant tells us nothing about the differences in heme orientation stability between the NP1/NP4 and NP2/NP3 pairs of proteins.

The NP2(D1A,Belt) mutant in which “Belt” = F42A shows a large predominance of the A heme orientation, A:B ~10:1, but with the seating of both A and B being very close to that expected for having the axial ligand plane oriented at exactly 135°. In support of the assignment of A as the major isomer, we note that the two overlapped methyl resonances at 68 ppm are at a very similar chemical shift to that of the small methyl resonance of the parent NP2(D1A) complex (Figure 3), while the small resonance at ~64.5 ppm is at a very similar chemical shift to that of the large resonance of the two overlapped methyl resonances at 65 ppm. At the same time, the small, broad resonance at 54 ppm for NP2(D1A) is at a very similar chemical shift as the large methyl resonance of NP2(D1A,F42A) assigned to the 3M group of that mutant. We also note that the large His57 β-CH resonance is at higher chemical shift, 21.5 ppm, with the small resonance being at 20.0 ppm. This is the opposite order of that found in all cases of (M0)NP2 mutants where separate His57 β-CH resonances have been observed, in which the B heme orientation is more abundant than the A [38]. Assignment of the heme resonances of high-spin NP2(D1A,F42A) is shown in Figure 5. This material comes from the assignment of the low-spin NMeIm complex, Supporting Information Figure S7, and the STD spectra which connect the low-spin assignments to the high-spin assignments, Supporting Information Figure S8. Thus we see that the mutation F42A makes the A heme orientation very highly favored, the only single mutant of (M0)NP2 or NP2(D1A) besides (M0)NP2(V24E) [37, 66, 67] in which the A heme orientation is favored, and, as was the case in that former study, mutation of only one residue is required to create this opposite preference for heme orientation. Thus position 42 of NP2 is extremely important in determining the A:B heme orientation ratio.

Figure 5.

Mutants aimed at re-creating, in a stepwise manner, the heme environment of NP4, and their ¹H NMR spectra in the high-spin Fe(III) state. For NP2(D1A,I120T) the methyl resonances of A are identified in blue lettering, whereas those of B are lettered in red.

Positions 42 and 106 for NP2 and the corresponding positions 44 and 107 for NP1 and NP4 are quite interesting, in that, as shown in Figure 1, there are residues at the positions 42/44 and at 106/107 which are the reciprocal of each other, with the 42/44 residue being mainly below and the 106/107 residue being mainly above the mean plane of the heme. NP2 has F42 and L106, while NP1 and NP4 have L44 and F107; in each case the larger F is paired with the smaller L, as shown in Figure 1, such that the amount of space utilized by the combination would seem to be fairly constant with respect to their effect on the orientation of the heme. We have already prepared and investigated in detail one of the two non-complementary mutants, NP1(L44F), in which two phenyl groups are facing and probably interfering with each other [38]. The ¹H NMR spectrum of the high-spin form of the non-complementary mutant NP1(L44F) was found to exhibit chemical exchange among the heme methyl resonances and among the propionate α-CH resonances [36, 38]. Thus, it appeared that both of the heme orientations, while present, cannot achieve stable seatings with the heme in either A or B orientation in this mutant, and were each changing their heme seating on the NMR timescale. Therefore, we did not prepare the other non-complementary mutant, NP2(L106F), either previously, or in this work.

We did, however, prepare a triple mutant of NP2(D1A), which was designed to mimic the heme pocket of NP1, or better (because of the N-terminus), of NP4, both of which have ~1:1 equilibrium ratios of A:B. The 1D ¹H NMR spectrum of this high-spin triple mutant, the aqua complex of NP2(D1A,F42L,L106F,I120T), is included in Figure 3, and is also shown, together with those of NP2(D1A), NP2(D1A,F42A), and NP2(D1A,I120T) in Figure 5, which shows the changes in the spectra with each mutation, albeit F42L in the triple mutant of NP2(D1A). By detailed investigation of this triple mutant of NP2(D1A), described below, as both the high- and the low-spin complex, we have confirmed that the heme orientation is very nearly all A, about 25:1 A:B, unlike the high-spin wild-type NP4 aqua complex, which has A:B ~1:1. Thus, this NP2(D1A) triple mutant has far over-corrected for the preference of the B heme orientation of NP2(D1A), an approximate change in the equilibrium constant from 1:14 to 25:1, a factor of 350. This overcompensation is likely a result of the fact that the fit of the heme inside NP2 is much tighter than inside NP4 or NP1, as can be seen by the much more severe ruffling of the heme for NP2 as compared to NP4 or NP1, as shown in Supporting Information Table S1. Thus, when the residues native to NP2 are replaced by those of the smaller side chains present in NP4, the result is a strong overcompensation and stabilization of the A heme orientation over B.

The heme resonances of the high-spin aqua complex of NP2(D1A,F42L,L106F,I120T) were assigned via saturation transfer experiments (shown in Supporting Information Figure S10 and S11) carried out on solutions which contained about 0.25 equivalent NMeIm, using the assignments of the low-spin NMeIm complex shown in Supporting Information Figure S9. In addition, a WEFT-NOESY spectrum confirmed the α-CH₂ assignments of the 6- and 7-propionates of the pure high-spin complex (Supporting Information Figure S12). Unique features of the 1D ¹H NMR spectrum of the high-spin form of this mutant include: a) 2-vinyl-α-H, at 55.2 ppm, has a larger chemical shift than 3M, and the same as the 55.2 ppm chemical shift of 4-vinyl in NP2(D1A,Y104A) discussed above; b) 4-vinyl-α-H has a larger chemical shift (42.5 ppm) than in many other A heme orientation NP2 mutants; c) the His57 β-CH resonance at 20.2 ppm is accompanied by a very small resonance at about 18.8 ppm that arises from the B heme orientation.

NMR Characterization of the Low-Spin Ligand Complexes of NP2(D1A) Belt Mutants

The 1D and WEFT-NOESY spectra of NP2(D1A,I120T)-NMeIm, NP2(D1A,Y104A)-NMeIm, NP2(D1A,F42A)-NMeIm, and NP2(D1A,F42L,L106F,I120T)-NMeIm are shown in Supporting Information Figures S2, S5, S7 and S9, respectively. As can be seen, two of these mutants show predominantly the A heme orientation, with ratios of A:B of about 10:1 and 25:1, respectively for NP2(D1A,F42A)-NMeIm and NP2(D1A,F42L,L106F,I120T)-NMeIm. The most difficult spectrum to assign was that of NP2(D1A,I120T)-NMeIm, because of having both A and B heme orientations present in equal abundance. While the 2Hα proton of B could be assigned, the 2Hα proton of A eluded assignment. The reverse was true for the 4Hα, where 4Hα and β of A could be assigned but 4Hα and β of B could not.

For NP2(D1A,Y104A)-NMeIm, for which the HMQC and WEFT-NOESY spectra are shown in Supporting Information Figure S5, we see that, as for the HS form of this mutant, the chemical shift of the 4Hα resonance is at an unusual chemical shift of 23.7 ppm, whereas it is usually found at about 6 ppm for the B heme orientation (6.4 ppm for NP2(D1A)-NMeIm, Table 2) [35]. The much larger chemical shift of 4Hα for the Y104A mutants of NP2(D1A) and (ΔM0)NP2 suggest a much larger spin density at that position, which, for the B heme orientation, is right at the site of the Y104A mutation. The very strong NOEs between 4Hα and 5M and between 4Hβ at 0.47 ppm and 3M, however, confirm that the vinyl group is still positioned more-or-less as seen in Supporting Information Figure S4, with 4Hα pointing toward 5M, albeit with the vinyl group fully in the plane of the heme. The chemical shifts and assignments of all complexes are presented in Tables 2 and 3, where the parent complex, NP2(D1A)-NMeIm [35] is also included in Table 2.

Table 2.

¹H and ¹³C chemical shifts of the NP2(D1A)-NMeIm at 32 °C, and NP2(D1A,I120T)-NMeIm complexes at 30 °C.

Heme	NP2(D1A)-NMeIm				NP2(D1A,I120T)-NMeIm
	A [ref 35]	[This work, 25.0 °C]	B [ref 35]	[This work, 25.0 °C]	A		B
A:B ratio at Equilibrium	1		14		1		1
Substituent	1H (ppm)	13C (ppm)	1H (ppm)	13C (ppm)	1H (ppm)	13C (ppm)	1H (ppm)	13C (ppm)
1M	−0.2	−	13.6	−23	1.27	−3.4	12.75	−19.4
3M	26.6	−51	13.8	−37	24.02	−49.4	14.95	−36.8
5M	1.1	−6	15.0	−31	3.57	−5.2	13.72	−26.3
8M	14.8	−31	1.7	−4	11.76	−22.3	1.98	−3.5
2α	12.0	55	22.6	−	−	−	21.17	−
2β	−4.3, −5.4	−	−5.3, −5.3	−	−4.1, −5.04	187.8	−5.47, −5.62	217.2
4α	10.6	75	6.4	52	8.69	83.1	−	−
4β	1.2, 0.9	126	1.2, 0.6	126	1.51, −0.06	153.1	−	−
6α	12.9, 12.1	−44	13.5, 13.0	−28	10.65, 5.3	−29.0	13.35, 12.61	−40.4
6β	−0.9, −2.1	136	−0.6, −2.0	134	−2.42, −2.9	114.3	−1.1, −2.25	134.5
7α	9.7, 5.1	−45	8.7, 4.4	−28	12.97, 11	−38.1	8.78, 4.08	−24.8
7β	−2.4, −2.9	108	−2.5, −3.1	103	−1.51, −2.64	129.1	−2.49, −3.04	105.2
m-α	−2.1	−	−1.6	−	−	−	−2.08	−
m-β	−	−	7.3	−	9.7	−	7.18	−
m-γ	−2.7	−	−3.1	−	−3.54	−	−3.33	−
m-δ	7.5	−	9.9	−	7.94	−	9.23	−
H57 α-CH	8.4	70	8.4	70	8.27	72.7	8.45	71.7
H57 β-CH	12.2, 6.2	18	12.2, 6.2	18	12.28, 6.33	20.6	12.49, 6.25	21
M order	3>8>5>1		5>3>1>8		3>8>5>1		3>5>1>8
M Av. Shift, ppm	10.0	−	11.03	−23.8	10.16	−20.1	10.85	−21.5
M spread, ppm	26.8	−	13.30	33	22.75	46.0	12.97	33.3
δ5M – δ1M, ppm	1.30		2.30		2.30		0.97
Angle	111–112°		161–162°		~117–118°		157–158°
Δφ	23–24°		26°		17–18°		21–23°

Table 3.

¹H and ¹³C chemical shifts of the NP2(D1A,Y104A)-NMeIm, NP2(D1A,F42A)-NMeIm and NP2(D1A,F42L,L106F,I120T)-NMeIm complexes at 30 °C (major A or B heme orientation as marked).

Heme	NP2(D1A,Y104A)-NMeIm B		NP2(D1A,F42A)-NMeIm A		NP2(D1A,F42L,L106F,I120T)-NMeIm A
Substituent	1H (ppm)	13C (ppm)	1H (ppm)	13C (ppm)	1H (ppm)	13C (ppm)
1M	12.84	−19.6	−0.61	1.1	2.24	−3.4
3M	15.36	−35.7	27.17	−54.0	23.85	−49.7
5M	13.58	−25.2	1.59	−2.6	3.91	−5.2
8M	2.76	−5.6	14.28	−26.6	10.42	−20.1
2α	21.71	29.5	11.91	58.5	19.49	55.9
2β	−4.88, −4.88	214.2	−4.53, −4.87	175.3	−2.34, −2.93	118.8
4α	23.73	49.9	10.14	77.5	7.40	86.9
4β	1.18, 0.47	135.6	1.12, −0.52	158.8	1.79, 0.41	148.9
6α	17.29, 10.55	−40.5	9.72, 4.37	−26.9	11.85, 6.04	−31.6
6β	−0.34, −1.82	134.5	−2.64, −2.97	109.5	−2.45, −3.70	188.2
7α	9.77, 4.35	−26.0	14.21, 10.88	−37.8	13.23, 9.62	−36.5
7β	−2.48, −3.15	107.4	−1.23, −2.52	128.1	−1.64, −2.97	125.0
m-α	−2.27	52.1	−2.33	54.2	−0.71	−
m-β	7.47	22.0	10.02	−	10.27	17.2
m-γ	−3.67	45.1	−3.28	45.1	−3.83	−
m-δ	8.65	19.6	7.62	28.5	8.43	−
H57 α-CH	8.58	71.9	8.24	72.7	8.23	73.3
H57 β-CH	13.16, 6.88	21.8	6.12, 12.77	19.5	12.54, 6.32	19.6
Ile120 CδH3	−2.44	13.7	−2.26	15.1	−	−
M order	3>5>1>8		3>8>5>1		3>8>5>1
M Av. Shift, ppm	11.14	−21.5	10.61	−20.5	10.11	−19.6
M spread, ppm	12.60	30.1	27.78	55.1	21.61	46.3
δ5M – δ1M, ppm	0.74		2.20		1.60
Angle	153°		112–113°		~121°
Δφ	20°		22–23°		~13°

For the low-spin complex of the triple mutant of NP2(D1A) bound to NMeIm, the complex exhibits one major heme orientation, that being the A orientation; A:B ~25:1, as shown in Supporting Information Figure S9, as compared to 1:14 for NP2(D1A), a change in K_eq from 0.071 to 25, a factor of 350. The heme of the A orientation rotates clockwise by 2–3° to remove 2-vinyl from being near L42 and F106, and this 2-vinyl has its Hα resonance at the highest chemical shift observed thus far for this heme substituent in any NP2 mutant (55 ppm), a larger chemical shift than for 3M (52.3 ppm). For NP2(D1A) the 2Hα chemical shift is 44.8 ppm, and for most other mutants of Figure 3 it is at 42–45 ppm, at 45.3 ppm for NP2(D1A,F42A) and 55.2 ppm for NP2(D1A,F42L,L106F,I120T), while for NP2(D1A,Y104A) and (ΔM0)NP2(Y104A) it is at 38.2 ppm, and 38.0 ppm for NP2(D1A,Y85A). In contrast, the 4Hα, which is usually at 33–41 ppm, is found at 55.2 ppm for NP2(D1A,Y104A) and (ΔM0)NP2(Y104A), and fairly close at ~51.7 ppm for NP2(D1A,Y85A), suggesting that removal of the large tyrosyl side chains of each of these mutants forces the 4-vinyl group to be in much greater conjugation with the heme π system of the high-spin complexes. The reverse is true for the triple mutant of NP2(D1A), which has A heme orientation and has 2Hα at 55.2 ppm and 4Hα at 42.5 ppm. Since the triple mutant of NP2(D1A) has major heme orientation A, while the Y104A and Y85A mutants have B, it is unlikely that the chemical shifts of 2Hα or 4Hα are related to heme orientation. Rather, a larger chemical shift for either vinyl Hα is consistent with greater spin delocalization to that vinyl group, which is consistent with a more in-plane position of the vinyl side chain. Since being coplanar with the heme is a higher-energy state for the vinyls of protohemin, something must be forcing the 4-vinyl of Y104A mutants to be coplanar with the heme. Without a crystal structure of this mutant it is difficult to determine which protein side chain is involved, since it is clear that there could have been some major changes in packing of the side chains near 4-vinyl and 3-methyl in these mutants.

The NMeIm complex of NP2(D1A,F42L,L106F,I120T) has an extremely well-resolved NMR spectrum that does not exhibit any chemical exchange derived from two different environments for a particular resonance. Three well-resolved protein methyl resonances are observed at negative chemical shifts in the natural abundance ¹H{¹³C} HMQC spectrum, at ¹H chemical shifts of −0.7, −1.3 and −2.8 ppm, all with ¹³C shifts of ~+20 ppm, as seen in Supporting Information Figure S9. The first two likely arise from the Cδ1 and Cδ2 methyls of L132, both of which give cross peaks to 3M, but the third has not been assigned.

Summary and Conclusions

The NP2 proteins with N-termini ΔM0 and D1A continue to show extremely similar 1D and 2D NMR spectra, as reported previously [40], and can be investigated interchangeably. Ile120-δ-CH₃ is very close to the heme, and together with the cavity structure, defines the heme orientation (A:B ratio) inside the heme cavity, so that the I120T mutants of (ΔM0)NP2 and NP2(D1A) provide essentially equal stability for the A and B heme orientations. The mutation F42A reverses the heme orientation to A:B ~10:1. The triple mutant of NP2(D1A) vastly overcorrects for the approximately equal stability of the A and B orientations of the I120T mutants to create a A:B ratio of approximately 25:1. And finally, removal of a phenol side chain of (ΔM0)NP2 or NP2(D1A) from Y104 or Y85, replacing it with a proton (methyl of alanine vs. CH₂ of tyrosine), or by creating the NP2(D1A) triple mutant, allows 4Hα for B of Y104A mutants or 2Hα for A of the triple mutant to have a larger chemical shift than 3M, suggesting that the vinyl group is in as full conjugation with the heme π-system as possible. Thus, the effects of the vinyl groups, with just two extra carbons than would be present in the “symmetrical hemin,” 2,4-dimethyldeuterohemin, have a number of important effects on the NMR spectra of NP2, where the heme seems to fit more tightly into the calyx than is true of nitrophorins 1 and 4.