A proton NMR investigation of the influence of distal glutamine on structural and dynamic properties of elephant metmyoglobin.

The proton NMR spectra of metmyoglobin from the Asian elephant, which has the replacement of glutamine for the usual distal histidine, are reported and analyzed. In the low pH region, we detect two interconvertible forms of the met-aquo-protein whose relative stabilities are independent of pH, but depend strongly on both temperature and solvent isotope composition. As the pH is raised, both species convert to the met-hydroxy form, as found for other myoglobins. The temperature dependence of the heme methyl shifts for both acidic protein forms indicates essentially high spin character for the iron, and the mean heme methyl shifts are interpreted as indicating one form with a very slightly weaker, and the other with a significantly stronger, axial ligand field than for the unique sperm whale met-aquo-myoglobin. The thermodynamic data for the equilibrium between the two species are consistent with differences of one hydrogen bond between coordinated water and the distal glutamine. Models are proposed where one form of the protein has not only the glutamine carboxyl oxygen acting as a hydrogen-bond acceptor, but also the amine group. We conclude that a distal glutamine can act both as a stronger and as a weaker hydrogen-bond acceptor towards coordinated water than the usual distal histidine. The relative rates of conversion of the two met-aquo-myoglobin forms to MetMbOH is found to be consistent with the proposed structures for the two forms.

The proton NMR spectra of metmyoglobin from the Asian elephant, which has the replacement of glutamine for the usual distal histidine, are reported and analyzed. In the low pH region, we detect two interconvertible forms of the met-aquo-protein whose relative stabilities are independent of pH, but depend strongly on both temperature and solvent isotope composition. As the pH is raised, both species convert to the met-hydroxy form, as found for other myoglobins. The temperature dependence of the heme methyl shifts for both acidic protein forms indicates essentially high spin character for the iron, and the mean heme methyl shifts are interpreted as indicating one form with a very slightly weaker, and the other with a significantly stronger, axial ligand field than for the unique sperm whale met-aquo-myoglobin. The thermodynamic data for the equilibrium between the two species are consistent with differences of one hydrogen bond between coordinated water and the distal glutamine. Models are proposed where one form of the protein has not only the glutamine carboxyl oxygen acting as a hydrogenbond acceptor, but also the amine group. We conclude that a distal glutamine can act both as a stronger and as a weaker hydrogen-bond acceptor towards coordinated water than the usual distal histidine. The relative rates of conversion of the two met-aquo-myoglobin forms to MetMbOH is found to be consistent with the proposed structures for the two forms.
Current hypotheses regarding the structure-function relationships in myoglobin and hemoglobin assign an important role to the distal residue in stabilizing the iron-bound ligand (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). The vast majority of myoglobins and hemoglobins have a histidine as the distal residue which is thought to be crucial for proper function for this class of proteins. The various roles assigned to the distal histidyl imidazole include acting as a hydrogen-bond donor to the bound ligand in oxyand met-cyano proteins (Refs. 11 and 12, respectively), a hydrogen-bond acceptor towards coordinated water in metaquo-systems (13), (Le. Fig. 1, C ) , an electron donor towards heme bound carbonyls (14), and as a dynamic trap door (15) * This research was supported by grants from the National Institutes of Health, HL-16087, and the National Science Foundation, DEB-80-2116 and PCM 7717644. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ TO whom correspondence should be addressed.
which contrls entry and exit into the isolated heme pocket in myoglobin (16) and hemoglobin (3). While there exists a number of monomeric oxygen binding hemoproteins without a distal histidine (e.g. aplysia (171, Chironomus thummi thummi (18), Glycera (6)), comparison of their structure and properties with the more common distal histidine containing myoglobin and hemoglobin, in terms of difference in distal interactions, is complicated by the large number of important nonconserved amino acid substitutions, additions and/or deletions. It has been shown (19)(20)(21) recently, however, that Mb' from the Asian elephant has a distal glutamine substituted for the more common histidine, and exhibits only three other nonconserved amino acid substitutions which are all on the protein surface and hence likely functionally irrelevant. Elephant Mb has been shown to exhibit an oxygen affinity very similar to that of other myoglobins but a significantly reduced susceptibility to autoxidation (20). An acid + alkaline transition characteristic of distal histidine containing Mbs has also been found for elephant MetMb using optical spectroscopy (21), although with a reduced pK value. While structural consideration of a glutamine substituted for a histidine with a C, at the same position as in the x-ray structure of sperm whale Mb indicates that glutamine can approach the iron closer than histidine (20), ESR studies on elephant nitrosyl Mb indicate (21) a diminished interaction of the NO with the distal residue as compared to the same form of sperm whale Mb (21).
Proton NMR spectroscopy provides a particularly sensitive technique for monitoring interaction between heme and distal amino acid residues (22)(23)(24)(25)(26)(27)(28). In paramagnetic forms of the proteins such interactions modify the hyperfine shifts (29) experienced by the heme substituents, and in certain protein forms such as met-cyano-Mb, it is possible to resolve signals from the distal residue whose hyperfine shift and relaxation properties give direct information on the position of the distal residue (12,23). In this report we consider the 'H NMR properties of elephant met-aquo-Mb and compare them with those of the well characterized NMR properties of the analogous form of sperm whale Mb (24,26). We present evidence that elephant MetMb at low pH exists in two distinct but interconvertible forms previously undetected by visible spectroscopy (21), and show that the spectral parameters suggest that the two species differ in the extent of interaction of the coordinated water with the distal glutamine, showing both weaker and stronger interactions than in sperm whale MetMb. acting solely as a hydrogen-bond acceptor via its carbonyl group; B, glutamine acting as a hydrogen-bond acceptor with its carbonyl group as well as its amine nitrogen; C, histidine serving as a hydrogen-bond acceptor with the ring nitrogen.

EXPERIMENTAL PROCEDURES
Asian elephant Mb was isolated from skeletal muscle under CO as described earlier (20). The Met form was prepared by flash photolysing off the CO and oxidizing the protein with 2 eq of potassium ferricyanide and passing through a Sephadex G-25 column equilibrated with 0.02 M NaCl solution. The protein was eluted with 0.001 M NaOH, and the eluent was concentrated in an Amicon micro cell, and lyophilized. A typical NMR sample consisted of dissolving 30 mg of the lyophilized protein in 0.5 ml of 'HZ0 or 90% HZO, 10% 'HzO solution containing 0.2 M NaC1. The pH was adjusted using 0.2 M 'HCl or 0.2 M NaO'H. The pH was measured in the NMR tube using an Ingold micro combination electrode and a Beckman Model 3500 pH meter, and is uncorrected for the isotope effect.
'H NMR spectra were collected on a Nicolet 360 instrument operating at a proton frequency of 360.067 MHz using quadrature phase detection. A typical spectrum was obtained by presaturating water with a 150-ms decoupler nonphase selective pulse and computer averaging 5000 to 8000 transients of 4096 points over a band width of 40 khz. The signal/noise was improved by apodizing the free induction decays which introduced 60 Hz line broadening; account was taken of the artificial line broadening in all calculations. The chemical shifts were referenced to the residual solvent signal, which, in turn was calibrated against 2,2-dimethyl-2-silapentane-5-sulfonate. The line widths and areas of peaks were measured by using the NTCCAP curve fitting program available on the Nicolet 1180 Data System.

RESULTS
The influence of pH on the 360 MHz 'H NMR spectra of elephant MetMbH20 is illustrated in Fig. 2 Raising the pH has no effect on the position, relative intensities, and line widths of the MetMb peaks, although all resonances lose intensity while a new set of resonances (labeled Ci) appear. This species, identified as MetMbOH, has a spectrum very similar to that of sperm whale MetMbOH (24) (Fig. 2F).
Two subsets of resonances in elephant acidic MetMb are readily identified, Ai, Bi, on the basis of altering the relative population of the two species. The spectra of MetMbHzO in HzO and 'H20 at 5 "C are compared in Fig. 3, A and B. It is clear that, while the relative intensities of the various signals within a subset (Ai or Bi) remain unchanged, the Ai peaks are more intense relative to the Bi peak in H20 than in 'H'O.
Moreover, when the temperature is raised for either sample, the Ai peaks gain intensity at the expense of the Bi subset, as illustrated for the 'HZ0 solution in Fig. 3 and 'H20 are given in Fig. 4 and the thermodynamic parameters are listed in Table I.
The variable temperature data for the heme methyl resonance of each species are presented in Fig. 5 in the form of a Curie plot (29). The data lie on straight lines with apparent intercepts at T" = 0, similar to those found for sperm whale MetMbH20 (26). The shifts at 25 "C for the two sets of methyl peaks for elephant MetMbH,O are compared in Table I1 with similar data for sperm whale MetMbH20 (26).
In addition to the shifts presented in Fig. 5, the resonances of both Ai and Bi exhibit line broadening as the temperature is raised. The influence of temperature on the line width of peak AI is shown in Fig. 6. The decrease in line width with increasing temperature on the right is indicative of normal paramagnetic relaxation (30). The broadening experienced at higher temperature reflects exchange contributions in the NMR slow exchange limit, which can be analyzed via the standard equation ( where and 62 are the line width in the presence and absence of exchange for peak A, respectively; a similar equation holds for resonance Bi. Although similar line broadening is also detected for the B1 peaks at elevated temperatures, the lower intensities and poorer resolution precluded a quantitative analysis. The value of kAB derived from Equation 2, together with the estimated kBA using the relation K = kAB/ kgA are included in Table I. Thus, the exchange broadening at higher temperature must involve the interconversion between the A and B species. The exchange broadening of A, is found to be essentially independent of pH in the range 5-8. However, as species C (MetMbOH), is appreciably populated, the Ai resonances selectively experience additional line broadening, as illustrated in the variable temperature NMR traces at pH 9.0 in Fig. 7. Peaks Ai clearly broaden much more extensively than peaks Bi; in fact, peak B1 exhibits essentially the same line width at high temperature in the absence of C (Le. pH 6). A plot of the line width of peak A in the presence of species C is included in Fig. 6. This excess line broadening of the Ai resonances a t alkaline pH in the presence of MetMbOH must represent the additional slow exchange contribution (31) due to the interconversion between A and C as in reaction 3.

A-B k m
The absence of such excess broadening for Bi peaks dictates that the A + C interconversion is appreciably faster than the B + C interconversion (with the A + C rate also faster than the A + B rate). The values estimated at 35 "C are kAC -(16 k 5) X 10' s-'; k g~ -(0.5 f 0.1) X 10' s-'. kCA and kcB are not determinable due to the poor resolution of peaks for species C.

DISCUSSION
Equilibrium between Two Met-aquo-species-The optical spectrum of elephant metmyoglobin has been studied (21) as a function of pH and shown to be consistent with a simple equilibrium between acidic (met-aquo) and alkaline (methydroxy) forms, as is the case of sperm whale or human metmyoglobin, except that the pK (8.5) is lower by 0.4 unit in the elephant protein. The present NMR data yield the first direct evidence for two distinct species in the acidic pH region. The failure to detect both species by optical spectroscopy can probably be traced to the fact that the equilibrium between the two acidic forms is independent of pH.

TABLE I Thermodynamic and kinetic data for A $ B equilibrium in elephant met-quo-myoglobin
The temperature dependence of the methyl hyperfine shifts (Fig. 3)  to be assumed to be predominantly high spin (32). The average heme-methyl shifts for species A is slightly larger than that reported for sperm whale MetMbH20 (Table I), while the average shift for species B is quite a bit smaller. Five-coordinate high spin iron(II1) is known to exhibit significantly smaller average heme methyl hyperfine shifts in models (33). However, such five-coordinate models also exhibit a characteristic upfield (-40 ppm) meso-H shift (33) which cannot be detected for elephant MetMbH20 under conditions where the same resonance can be resolved downfield in sperm whale MetMbH,O (26). Hence, we conclude that both A and B must  Average 61.1 Data taken from Ref. 26. be six coordinate. In such species the average heme methyl shift can be expected to reflect the strength of the axial field, where the cis effect (34) predicts a decrease in iron-porphyrin u bonding (and hence, decreased heme methyl shifts), as the axial field strength increases (35).
On the basis of the closer similarity of the average as well as the spread of the heme methyl shifts to those of sperm whale MetMbHzO, we assign A the structure A in Fig. 1, where the glutamine NH acts as a hydrogen-bond donor similar to the histidyl imidazole in sperm whale MetMbH20 (Fig. 1C). On the basis of the slightly larger average heme methyl shifts for A, we conclude (34, 35) that the hydrogenbonding interaction with glutamine in A is slightly weaker than in sperm whale MetMbHzO. A similar decrease in the interaction between coordinated NO and distal hydrogenbond donor for elephant relative to sperm whale myoglobin has been deduced for nitrosyl myoglobins (21).
The large isotope effect (factor of - , and C (MetMbOHj which have resonances labeled Ai, Bi, and Ci, respectively. As the temperature is raised, the Ai peaks (particularly note A l j broaden considerably more than the Bi peaks (note B,) when C is present, reflecting additional exchange contribution which dictates that the A + C rate is much faster than the 8 + C rate. equilibrium constant observed in H20 and 'H,O suggests a structural difference between the two species involving a hydrogen bond with the coordinated water (36). The larger K in *HzO indicates stronger or more extensive hydrogen bond- 20 ing in species B. The difference in AG for the process A -B in both solvents is also consistent with the presence of one additional hydrogen bonding species B (36). The negative AS indicates more restricted rotation or motion in species B.
One difference between glutamine and histidine as distal residues is that the former has two potential sites for simultaneous interaction with the coordinated water. While the resonance stabilization usually makes the O=C-NH2 fragment planar, (Fig. 1A), it seems plausible that hydrogen bond donation towards the amine portion could stabilize a structure such as that depicted in Fig. 1B. The loss of the partial resonance would be compensated by the newly formed hydrogen bond, and this hydrogen bond would be stronger in 'H20 than H,O. The addditional hydrogen bond in Fig. 1B would also cause the coordinated water to act as a stronger u donor. This increased axial field would account for the smaller heme methyl hyperfine shift via the well known cis effect (34, 35).
Thus, species B indicates that the coordinated water is capable of interacting more strongly with the distal glutamine than a distal histidine. We propose that this species can be represented by the structure in Fig. 1B. The ability of glutamine to provide two sites for interaction with a coordinated ligand has also been invoked to explain the lower pK for the acid + alkaline transition via preferential stabilization of the coordinated hydroxy ligand (21).
The absence of influence of pH on the A/B equilibrium is also consistent with an "intermolecular" equilibrium between structures involving solely the coordinated water and the distal glutamine. Increasing the pH leads to conversion to the MetMbOII form (peaks Ci), which appears to be very similar to sperm whale MetMbOH. The apparent pK -8.7 in 2H20 is consistent with that reported for elephant MetMbH,O in H20 on the basis of optical data (21). The optical data, however, failed to discriminate between the two contributing acidic forms of the protein.
The Dynamics of Interconversion-The pH-dependent line broadening of both the Ai and Bi resonances at low pH is indicative of kAB and ItsA, both being first order rate constants.
The additional line broadening a t alkaline pH in the presence of species C for the Ai but not the Bi resonances dictates kAC is much larger than ksc. In considering the proposed structures of species A and B (Fig. 1, A and B ) , it is clear that one water proton is exposed for attack by hydroxide for the proposed structure of species A, while both protons are involved in interacting with glutamine in the structure of B. Thus, the proposed structure of A and B provide a rationalization for the reduced liability of the water proton in species B relative to that in species A.
Thus, we conclude glutamine is capable of interacting strongly with the coordinated ligand in MetMb. Similar conclusions are indicated by the relaxation properties of exchangeable protons in the heme cavity of elephant met-cyano-Mb.2 Further NMR investigation of ligated forms of elephant myoglobin designed to provide further characterization of distal interactions are in progress in these laboratories.