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Originally published In Press as doi:10.1074/jbc.M401561200 on March 11, 2004

J. Biol. Chem., Vol. 279, Issue 22, 22944-22952, May 28, 2004
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Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding*

Karin Nienhaus{ddagger}, Jan M. Kriegl{ddagger}, and G. Ulrich Nienhaus{ddagger}§

From the {ddagger}Department of Biophysics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany and the §Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, February 12, 2004 , and in revised form, March 11, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4–11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Globins are small respiratory proteins that reversibly bind dioxygen and other small ligands at the central iron of a heme prosthetic group that is embedded in a highly conserved {alpha}-helical globin fold. They are widely distributed over many taxa, including unicellular eukaryotes, plants, fungi, and animals (1). Hemoglobin (Hb)1 and myoglobin (Mb) are the most prominent members of this protein family (2).

Recently, two new members have been discovered in the vertebrate globin family, cytoglobin, also known as histoglobin, and neuroglobin (Ngb) (3–6). Whereas cytoglobin is ubiquitously found in vertebrate tissue, Ngb is predominantly expressed in neuronal cells of the brain (4, 5, 7, 8). Phylogenetic analysis shows that Ngb is an ancient protein that existed long before the genes encoding Mb and Hb diverged. Nevertheless, it displays all key determinants of the globin fold (9). Ngb consists of a single chain of 151 amino acids and shares only 20–25% sequence identity with vertebrate Mb or Hb. Human and murine Ngb have 94% identical residues and are thus more similar than Hb and Mb in these species (77–85%) (5).

In the absence of an exogenous ligand, the heme iron is hexacoordinate in both ferric (Fe3+, met) and ferrous (Fe2+, deoxy) Ngb (5, 10–12), with His64 and His96 as axial ligands, as has been confirmed by recent x-ray structure analyses of human (13) and murine (14) met Ngb. Similar hexacoordinate globins have also been isolated from bacteria and unicellular eukaryotes (truncated hemoglobins (15, 16)) as well as from plants (nonsymbiotic hemoglobins (17–19)) and insects (20). The exogenous ligand replaces the endogenous one upon binding at the sixth coordination, and therefore, hexacoordination has been considered as a novel mechanism for regulating ligand binding affinity to the heme (13). The physiological role of hexacoordinate globins has not yet been clarified. For Ngb, the low expression level and moderate oxygen affinity (half-saturation pressure P50 {approx} 2.0 torr (5)) suggests a function different from simple oxygen storage and transport (21). Recent studies have reported an up-regulation of Ngb under conditions of hypoxia or ischemia in vitro (21, 22) and in vivo (23), thus promoting neuronal survival. The induction of Ngb expression by hemin indicates that more than one signal transduction pathway is involved in the regulation of Ngb expression, because heme availability and hypoxia-induced pathways are controlled through different mechanisms (24). Moreover, ferric (but not O2-ligated ferrous) human Ngb interacts with the GDP-bound form of the {alpha} subunit of the heterotrimeric G protein, thereby preventing neuronal death (25). All of these results suggest that Ngb is a sensor responsive to oxidative stress.

To understand the function of Ngb in the vertebrate brain and that of hexacoordinate globins in general, a thorough investigation of the structure, dynamics, and ligand binding (equilibrium and kinetic) properties is a prerequisite. Fig. 1 shows the essential features of the active site of murine met Ngb, as determined at 105 K (14). The heme is inserted into the globin in two different orientations, as already suggested from NMR investigations by La Mar and co-workers (26), which also affects the axial residues His64 and His96. Structural heterogeneity in the distal heme pocket was also apparent from infrared spectroscopy on CO-ligated Ngb (27).



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  		FIG. 1.
Active site structure of murine Ngb (14). The heme and selected residues are depicted in dark and light gray for the major and minor heme conformer, respectively.

 
Kinetic studies have revealed extremely fast CO and O2 association to pentacoordinate deoxy Ngb and extremely slow binding to the hexacoordinate deoxy form. The latter process is governed by thermal dissociation of the His64 side chain from the sixth coordination (10, 12, 27). Additional processes involving other amino acid side chains and/or ligands were inferred from infrared and optical absorption (27) and Raman (28) measurements. To examine structural and dynamic aspects of ligand binding, we have prepared Ngb mutants H64L, K67L, and H64L/K67L. Here we present steady-state optical absorption spectroscopy on the deoxy, met, and CO-ligated forms and time-resolved spectroscopy on the CO-ligated proteins. Measurements over a wide range of pH enabled us to examine the competition of endogenous and exogenous ligands for the sixth heme coordination as a function of the protonation states of the residues in the vicinity of the active site.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—Murine Ngb (wt) was produced in Escherichia coli strain BL21 (DE3)pLys by using the pET3a expression plasmid (27). Mutants H64L and K67L and double mutant H64L/K67L were constructed using the QuikChange mutagenesis kit (Stratagene) following the manufacturer's protocol. Custom designed primers were purchased from MWG (MWG-Biotech GmbH, Ebersberg, Germany). The mutants were expressed and purified as described for the wt protein (27).

Sample Preparation—Optical absorption spectra and flash photolysis kinetics at ambient temperature were measured on dilute aqueous samples, prepared by dissolving lyophilized protein in 100 mM sodium phosphate/citrate (pH 4–6), sodium phosphate (pH 6.2–8.6), and sodium carbonate buffer (pH > 8.6) to a final concentration of ~10 µM. For cryospectroscopy, the protein was dissolved in 75% glycerol, 25% potassium phosphate buffer (v/v). To produce NgbCO samples, the solutions were equilibrated with 0.05 or 1 atm CO partial pressure under anaerobic conditions, and a 10-fold excess of sodium dithionite was subsequently added. Six-coordinate deoxy Ngb samples were prepared by adding dithionite solution anaerobically to ferric Ngb solutions after thorough purging with N2 gas.

Absorption Spectroscopy—Spectra at ambient temperature were taken with a Cary 1 E spectrometer (Varian, Darmstadt, Germany) at a resolution of 1 nm. Low temperature spectra were recorded on an OLIS-modified Cary 14 spectrometer (On-Line Instrument Systems, Bogart, GA) at a resolution of 0.3 nm. The samples were cooled with a closed cycle helium cryostat (model 22, CTI Cryogenics, Mansfield, MA) equipped with a Lakeshore Cryotronics model 330 digital temperature controller. To observe photolysis-induced spectral changes, the samples were illuminated at low temperature for 10 min prior to data collection by using a frequency doubled Nd:YAG laser emitting ~300 mW at 532 nm (model Forte 530–300; Laser Quantum, Manchester, UK).

Flash Photolysis Experiments—In our home-built flash photolysis apparatus, photolysis was achieved by a saturating 6-ns (full width at half-maximum) pulse from a frequency doubled, Q-switched Nd:YAG laser (model Surelite II; Continuum, Santa Clara, CA). Light-induced absorbance changes were monitored in the Soret region using light from a tungsten source (model A 1010; Photon Technology International Inc. (PTI), Brunswick, NJ). The wavelength of the monitoring beam was adjusted by a monochromator. The transmitted light intensity was measured with a photomultiplier tube (model R5600U, Hamamatsu Corp., Middlesex, NJ) and recorded with a digital storage oscilloscope from 10 to 50 µs (model TDS 520; Tektronix, Wilsonville, OR) and a home-built logarithmic time base digitizer (Wondertoy II) from 2 µs to 100 s. Between 100 and 500 individual transients were averaged for each data set. For single-wavelength kinetics, the absorbance change was measured at 436 nm and normalized to 1 at the earliest times (30 ns). We denote the normalized absorbance change by N(t); it is the fraction of molecules that have not yet rebound a ligand at time t after the photolyzing flash.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Optical Absorption at Ambient Temperature—The spectra of Ngb mutant K67L in the different oxidation and ligation states of the heme iron are similar to those of wt Ngb (10). The two ({alpha} and {beta}) bands between 500 and 600 nm are indicative of hexacoordination of the heme iron in the oxidized (met) and reduced (deoxy) states without exogenous ligand as well as the CO-ligated state. The peak positions of the various bands are compiled in Table I. For the CO-ligated form, a small but significant spectral change with pH was noticed. With decreasing pH, the Soret ({gamma}) band of wt NgbCO (K67L NgbCO) redshifts from 415.6 to 416.6 (416.5 to 417.2) nm according to the Henderson-Hasselbalch relation (Fig. 2a). This relation gives the fractional populations of the protonated (acid) and deprotonated (base) species, here denoted by c+ and c0, as a function of pH.

((Eq. 1)
For the Henderson-Hasselbalch relation, which describes a single protonation, the parameter n equals 1; n > 1 signals a cooperative transition involving more than one protonation. The data in Fig. 2a show that a protonating group with a pK of 4.4 ± 0.1 (5.6 ± 0.1) resides in the vicinity of the heme. For the CO-ligated mutants with leucine at position 64, a similar shift of the Soret band was not observed.


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  		TABLE I
Peak positions of the absorption spectra of Ngb and mutants H64L, K67L, and H64L/K67L in different ligation and oxidation states for the different pH regions

 



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  		FIG. 2.
pH-dependent spectral changes of Ngb and mutants. a, the symbols show the positions of the Soret peak of wt and K67L NgbCO, plotted as functions of pH. b, absorption changes in the spectra of the H64L mutant. Closed symbols, ferric form at 409 nm; open symbols, ferrous form at 557 nm. The ordinate is scaled such that it represents the fraction of the deprotonated species. c, absorption changes in the spectra of ferric H64L/K67L. Closed symbols, changes in the absorption at 403 nm; open symbols, shift of the Soret maximum. The lines represent the best fits to the data using Equation 1, with Henderson-Hasselbalch relations (n = 1) except for the Soret shift data in c, which is a cooperative transition with n = 3.5.

 
In the absence of an exogenous ligand, replacing His64 by leucine results in substantial pH-dependent spectral changes in both the ferric and ferrous forms (Fig. 3, a and b). At low pH, the deoxy form of H64L Ngb is characterized by a broad Soret band, with a maximum at 422 nm and a pronounced shoulder at 436 nm (Fig. 3b). The second derivative spectrum in Fig. 3c displays two minima, clearly indicating that the Soret consists of two peaks. The spectral region between 500 and 600 nm exhibits a broad absorption feature approximately 550 nm. It contains multiple bands, as seen from the (most pronounced) minima in the second derivative spectra at 530, 560, and 571 nm (Fig. 3c). The spectra are pH-independent between pH 5 and 8. At more alkaline pH, they change dramatically (Fig. 3, b and c). The Soret band becomes much larger and narrower; the shoulder at 436 nm disappears completely. Moreover, two narrow and tall bands at 527 and 557 nm arise from the broad 550-nm absorption. These changes clearly signal the appearance of a new low spin heme iron species. In Fig. 2b, the absorbance change at 557 nm is plotted as a function of pH after rescaling from 0 to 1. The line through the data is the result of a nonlinear least squares fit of the data with a Henderson-Hasselbalch relation, yielding a pK of 9.9 ± 0.1. This behavior suggests that an amino acid near the active site deprotonates and binds to the heme iron.



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  		FIG. 3.
pH dependence of absorption spectra of Ngb mutants H64L and H64L/K67L. a and d, ferric forms, spectra expanded by a factor of 5 from 450 to 700 nm. b and e, ferrous forms, spectra expanded by a factor of 5 from 480 to 700 nm. e and f, second derivatives of the ferrous spectra in b and e at pH 6 (solid line) and 11 (dotted line), spectra expanded by a factor of 10 from 480 to 700 nm. The arrows indicate increasing pH.

 
At acidic pH, ferric Ngb H64L has a Soret peak at 403 nm (Fig. 3a). Above 500 nm, the spectrum exhibits the typical, multi-humped appearance of an aquomet globin species (2), with the charge-transfer band at 631 nm, which is characteristic of a high spin ferric heme iron (29). With increasing pH, the Soret peak shifts to the red by ~6 nm, and concomitantly, a double-humped spectrum appears above 500 nm, indicating that H2O at the sixth coordination is replaced by another ligand. Fig. 2b shows the scaled absorbance change at 409 nm in the Soret peak as a function of pH. The pH dependence is in excellent agreement with a Henderson-Hasselbalch relation with pK = 7.3 ± 0.1. Above pH 9, the absorption at 409 nm decreases slightly, which signals a protonation with pK {approx} 10. This is likely the same protonation that has already been much more obvious in the deoxy spectra.

The ferric double mutant H64L/K67L also shows the acidic/alkaline transition (Fig. 3d). From the change in absorbance at 403 nm, a pK of 8.8 ± 0.3 was determined (Fig. 2c). At pH > 9, a second transition manifests itself in a pronounced and rather abrupt shift of the Soret band from 407–421 nm (shown scaled from 0 to 1 in Fig. 2c). The fit with Equation 1 yielded a cooperativity parameter n = 3.5. The {alpha} and {beta} bands change very little, however (Fig. 3d). By contrast, the spectra of the ferrous species appear essentially pH-independent (Fig. 3e). The second derivative spectrum (Fig. 3f) only shows slight shifts of the bands with pH.

Optical Spectra at Cryogenic Temperature—After photodissociation of CO or other small, gaseous ligands at ambient temperature, a pentacoordinate deoxy Ngb forms that is converted within milliseconds to the hexacoordinate deoxy species by bond formation between the heme iron and the His64 imidazole (10, 27). At cryogenic temperature, however, large scale protein motions are frozen in (30, 31). Therefore, the His64 imidazole side chain is immobilized, and the CO rebinds to the pentacoordinate heme iron from the distal heme pocket. We have shown earlier that the enthalpy barrier for CO binding to pentacoordinate Ngb is extremely low in comparison with other heme proteins (27, 32). As a consequence, only at very low temperatures can a significant amount of pentacoordinate NgbCO photoproduct be trapped for longer periods of time after photolysis. Fig. 4a shows the Soret region of the NgbCO absorption spectrum before and after illumination at 10 K. Upon photodissociation, the intensity of the Soret peak at 414 nm decreases, concomitant with the appearance of the broad absorption of the pentacoordinate species with a maximum at 438 nm, as is evident from the difference spectrum. Essentially identical spectral changes are seen for mutant H64L (Fig. 4b), consistent with our expectation that His64 cannot bind to the sixth coordination in the native protein after photodissociation of CO at low temperatures.



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  		FIG. 4.
Trapping of pentacoordinate ferrous Ngb at cryogenic temperatures by continuous photolysis of ligated Ngb. a, NgbCO; b, H64L NgbCO. Solid and dashed lines represent spectra recorded, respectively, before and after illumination with light from a frequency-doubled Nd:YAG laser. Open circles, photolysis difference spectra. c, spectra of ferrous H64L measured at 290 K (dotted line) and 10 K (solid line) and at 10 K after illumination (dashed line). Open circles, photolysis difference spectrum. Closed circles, difference spectrum calculated from the spectra at 290 and 10 K. d, second derivatives of the spectra in c.

 
To further elucidate the complicated spectra of ferrous H64L Ngb at room temperature (Fig. 3b), we also performed cryospectroscopy on this species. The spectra in Fig. 4c were collected on a sample at pH 7.5. They show a pronounced and completely reversible temperature effect. With decreasing temperature, the peak at 438 nm gets weaker at the expense of the band at 418 nm. After laser illumination, this change is essentially reverted, which indicates that the band at 418 nm belongs to a hexacoordinate species with a small, photolyzable ligand at the sixth coordination, whereas the band at 438 nm belongs to pentacoordinate, high spin heme. Comparison of samples in the pH range 6–10 showed that the hexacoordinate species grows at the expense of the pentacoordinate form toward higher pH (data not shown). A third, rather weak feature at 428 nm is only visible in the better resolved spectra at 10 K, both before and after photolysis (Fig. 4, c and d). However, it is too weak to decide unambiguously from the data whether its fraction decreases or increases upon photolysis.

CO Binding after Photodissociation at Ambient Temperature—The ligand binding reaction in NgbCO after photodissociation is a complicated process that can best be followed from the time dependence of entire optical spectra. Fig. 5 shows a contour plot of the spectral changes that occur after photodissociation of wt NgbCO, pH 4, 0.05 bar CO, logarithmically plotted as a function of time between 30 ns and 10 s. Negative contours (dotted lines) on the left represent changes in the Soret band of NgbCO, and positive contours (solid lines) on the right represent changes in the Soret band of the species that occur upon CO photodissociation. A geminate phase caused by CO molecules that do not exit into the solvent after photodissociation has been observed at 275 K at times below 100 ns (27). At room temperature, this process is faster than 30 ns and thus outside of the time window of our instrument. A large fraction rebinds in a bimolecular process on the time scale of ~10–3 s, as is apparent from the decrease in the difference spectrum at that time. This process does not complete, however, but stalls for many orders of magnitude in time because the endogenous His64 binds to the heme iron, thus blocking the active site. Only after thermal dissociation of His64, which occurs on the 1-s time scale at pH 4, can the rest of the CO finally bind and the difference spectrum decay to 0. This interpretation is evident from the spectral changes in the Soret band of the photoproducts. The positive signal at early times is centered on ~432 nm but shifts to 425 nm because of the binding of His64 to the sixth coordination.



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  		FIG. 5.
Contour plot of the difference spectrum of wt Ngb after photolysis. The measurement was done at ambient temperature with protein dissolved in 0.1 M buffer, pH 4, equilibrated with 0.05 bar CO. Solid and dotted lines indicate positive and negative absorbance differences, respectively, with respect to the CO-ligated form. Contours are spaced logarithmically. The vertical line marks the default monitoring wavelength of 436 nm.

 
In most experiments, we take kinetic data only at a fixed wavelength, normally near the maximum of the pentacoordinate species at 436 nm (Fig. 5, vertical line). In Fig. 6, we compare CO rebinding in wt Ngb and its mutants with wt Mb and Mb H64L. All kinetic traces were taken on samples in 0.1 M buffer, pH 8, equilibrated with CO at a partial pressure of 0.05 bar, except for a single trace with wt NgbCO (open circles, 1 bar CO). In the time window of our experiments, the Ngb kinetics at 436 nm show two decays, characterized by apparent rate coefficients {lambda}1 and {lambda}2 and amplitudes N1 and N2. The first step in the kinetics at 1 bar is much faster than at 0.05 bar, as expected for bimolecular recombination of CO to the pentacoordinate deoxy form. The lines represent double-exponential fits to the data, and the resulting second order rate coefficients for CO binding to the pentacoordinate deoxy species are summarized in Table II. Note that the amplitudes in these traces do not represent the relative amounts of the two transient deoxy species because the first decaying species is measured near its absorption maximum, whereas the second, hexacoordinate species is measured in the wing of its absorption band (Fig. 5).



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  		FIG. 6.
Comparison of bimolecular rebinding of CO in Ngb and Mb. All of the proteins were dissolved in phosphate buffer solution, pH 8, and equilibrated with 0.05 bar CO (native Ngb also equilibrated with 1 bar CO (open circles)). The solid lines represent fits with one or two exponentials, respectively.

 


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  		TABLE II
Bimolecular rate coefficients

Shown are the bimolecular rate coefficients {lambda}'CO (in µM–1 s–1) for rebinding of CO from the solvent to Ngb and selected heme proteins, determined at 293 K in 0.1 M sodium phosphate buffer (pH 8) from 0.05 and 1 bar CO data. The solubility of CO in water at 293 K and 1 bar CO was ~1 mM (50).

 
CO binding in mutant K67L is similar to wt Ngb, slightly faster but also biphasic, with two well separated steps. By contrast, mutants H64L and H64L/K67L yield essentially monophasic kinetic traces. Their rate coefficients are ~4–5-fold larger than the one of the wild type protein (Table II). Consistent with the kinetic behavior at low temperature (27), the association rate for CO binding to wild type Ngb at ambient temperature is ~100 times higher compared with wild type sperm whale Mb and even two to three times higher than in Mb H64L, which binds ligands the fastest among all Mb mutants studied so far (33).

pH Dependence of CO Binding—The titration curves in Fig. 2 and the absorption spectra in Fig. 3 reveal a strong pH dependence of the ligation states in Ngb and its mutants. Therefore, we have also measured the absorbance changes of wt NgbCO and mutants at 436 nm after photodissociation at ambient temperature as a function of pH. Fig. 7 shows kinetic traces of wt NgbCO for two different CO concentrations in the solvent, obtained by equilibration with 1 bar (Fig. 7a) and 0.05 bar CO (Fig. 7b). The larger amplitude of the second step at 0.05 bar CO is evidence of the competition between CO and His64 for the sixth coordination. The slower bimolecular CO recombination at 0.05 bar gives the His64 imidazole side chain a greater probability of binding at the heme iron. The pH dependence of the kinetics is complicated. The bimolecular process slows with decreasing pH, the yield of the hexacoordinate deoxy species varies with pH in a complicated fashion, and the termination of the second step shifts to shorter times for acidic pH (pH ~4), which reflects destabilization of the bond between the heme iron and the endogenous His64 ligand. The kinetic traces of mutant K67L are similar to those of wt Ngb. Bimolecular recombination occurs on similar time scales and also slows toward low pH, and amplitude and termination of the second step are also pH-dependent.



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  		FIG. 7.
pH dependence of the flash photolysis kinetics of wt and H64L NgbCO. a, Ngb equilibrated with 1 bar. b, Ngb equilibrated with 0.05 bar CO. c, mutant H64L equilibrated with 0.05 bar CO.

 
NgbCO mutants H64L and H64L/K67L show monophasic recombination kinetics at 1 bar CO. Only when the concentration of CO above the sample is reduced to 0.05 bar or below, does a second process become apparent for mutant H64L. Fig. 7c shows a few select traces. The kinetics are pH-independent below pH 9. At pH > 9, CO association to the pentacoordinate species becomes faster with increasing pH, and a second, slower process occurs. In the double mutant H64L/K67L, we were unable to detect any pH-dependent changes in the kinetics between pH 5 and 11.

Fig. 8 shows apparent rate coefficients, {lambda}1, for the first step in the kinetics of wt Ngb and mutants K67L, H64L, and H64L/K67L between pH 4 and 11. At 1 bar of CO, endogenous ligand binding is small and thus {lambda}1 is essentially identical with the CO association rate coefficient. For wt NgbCO, there is a pronounced increase of the CO binding rate by a factor of ~4 toward higher pH values, well described by a Henderson-Hasselbalch relation with pK 6.0 ± 0.1. For mutant K67L, a similar behavior is observed, with a slightly larger amplitude of the transition and a shift to pK 6.3 ± 0.1. By contrast, a systematic variation of the rate with pH could not be detected in this pH range for mutants H64L and H64L/K67L. To observe the Henderson-Hasselbalch dependence for the overall rate, it is obvious that the protonated and unprotonated species must have different binding rates. Moreover, conformational fluctuations between them have to be faster than the reaction rate; otherwise, we would observe two separate kinetic species. The overall rate coefficient {lambda}(pH) is given by the linear combination shown in the following equation.

(Eq. 2)
The data in Fig. 8 yield excellent agreement with this relation. The missing pH dependence of the mutants lacking His64 suggests that this residue is responsible for the changes.



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  		FIG. 8.
pH dependence of the apparent rate coefficients for the bimolecular CO rebinding step of wt Ngb and mutants. The rates from exponential fits to the experimental data are represented by symbols. The solid lines show fits of the Henderson-Hasselbalch relation to the pH dependence. The dashed lines represent averages of the pH-independent apparent rate coefficients.

 
Fig. 9 shows a complicated pH dependence of the amplitude of the second step, N2, which is proportional to the yield of hexacoordinate Ngb after photodissociation, for wt NgbCO and mutant K67LCO at 0.05 and 1 bar CO. In addition to the overall increase of the yield of hexacoordinate deoxy Ngb with decreasing CO concentration mentioned earlier, there is up to 10-fold yield variation with pH. The increase in N2 of wt Ngb in the alkaline region (pH {approx} 10) is absent for the K67L mutant. In the acidic region, the hexacoordinate yield is low at pH 4, increases to a maximum at pH 6 for wt NgbCO, and afterward decreases again toward pH 8. The peak is shifted to slightly higher pH for the K67L mutant. Apparently, the maximum yield of hexacoordinate species coincides with the pK of the bimolecular CO association to the pentacoordinate species (Fig. 8).



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  		FIG. 9.
pH dependence of the amplitudes N2 of the second kinetic step. NgbCO (circles) and mutant K67L (squares) equilibrated with 1 bar (solid symbols) and 0.05 bar CO (open symbols) are shown. The lines are drawn to guide the eye.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ngb is a vertebrate globin with the canonical three-over-three globin fold (13, 14). Besides hexacoordination of the ferric and ferrous unligated species, it exhibits a few other remarkable structural features. A huge internal cavity with a volume of ~280 Å3 connects to a large tunnel through the matrix that may allow for easy ligand migration. Preliminary data indicate that the heme group shifts deeper into the heme crevice upon exogenous ligand binding. Whereas the structural data provide only a static picture, the results presented here provide some insights into the complex, time-dependent interactions among distal site residues His64, Lys67, the heme iron, and the exogenous ligand.

Equilibrium Conformations in the Presence of His64—The absorption spectra of ferric and ferrous murine Ngb and mutant K67L show hexacoordination over the entire pH range, with His64 and His96 as axial ligands (13, 14). In the CO-ligated state, the His64 imidazole is detached from the heme iron but still located within the distal heme pocket at physiological pH, as can be inferred from the Fourier transform infrared spectra of the CO stretching vibration (27). The pH-dependent Soret shifts with pK 4.4 and 5.6 in wt NgbCO and K67L NgbCO, respectively (Fig. 2a), are associated with His64 protonation because they are absent in the mutants lacking this residue. Further support for this assignment is provided by detailed studies of MbCO and its distal pocket mutants, which have proven that His64 protonation occurs in MbCO with pK 4.5 (34, 35). Yang and Phillips (36) have shown that, upon protonation, the His64 imidazole swings out of the hydrophobic distal heme pocket into the solvent to better solvate the charge. In NgbCO, the change in population of the CO stretching bands at 1940 and 1970 cm–1 in the Fourier transform infrared spectra with pK {approx} 4.4 suggests a similar scenario (27). These bands correspond, respectively, to the A1 and A0 bands in MbCO, which have been associated with the neutral and charged states of the His64 imidazole (34).

In ferric Ngb, His64 binds to the heme iron with its N{epsilon}2 atom. Bond formation impedes protonation, and consequently, a pH-dependent change in the absorbance spectra with pK {approx} 4.4 is absent. From acid denaturation studies of Mb it is known that the covalent bond between the proximal histidine and the heme iron breaks at pH < 3.5, with subsequent protonation of the histidine (37, 38). A similar scenario is likely for Ngb involving both axial histidines.

The difference in the pK of His64 between wt and K67L NgbCO indicates destabilization of the protonated form by ~6.7 kJ/mol in the presence of the charged lysine residue. In water at 298 K, the imidazole side chain of histidine has a pK of 6.14 (39, 40), with a preference of 4:1 for the proton at N{epsilon}2 instead of N{delta}1. In a protein, the pK values of protonating groups can vary drastically because of steric and charge interactions within their specific microenvironments. The pK of His64 in wt NgbCO is rather low for two reasons: (i) electrostatic interaction of His64 with the positive charge of Lys67 and (ii) low accessibility of the apolar protein interior for solvent. Lys67 forms a salt bridge to a heme propionate group, so that the Lys67 side chain acts as a stable barrier separating the distal pocket from the solvent. In the K67L mutant, the neutral leucine side chain cannot form a salt bridge to the heme propionates (14). Therefore, this modification is expected to facilitate access of solvent molecules to the distal heme pocket. The pK shift to 5.6 in this mutant, which is closer to the canonical pK of histidine, indeed suggests a more solvent-accessible environment of His64 in K67L NgbCO. Similar pK shifts of His64 have been observed in MbCO upon replacement of the neighboring residue Arg45 (33).

Equilibrium Conformations in the Absence of His64—In ferric wt Mb, the distal histidine stabilizes a water molecule at the sixth coordination (41). In ferric H64L Ngb, a sixth ligand is obvious from the spectra in Fig. 3a. The pH-dependent spectral changes reveal a protonation with pK 7.3 (Fig. 2b), suggesting heme ligation by H2O at acidic and OH– at alkaline pH. This acidic-alkaline transition, which has also been observed in human Ngb mutant H64V (28), is well known from many other ferric heme proteins (2), with pK values varying between 7.4 and 10.9. The pK in Chironomus insect Hbs is similar to the one observed in Ngb. Both globins also have a positively charged amino acid (arginine instead of lysine) at position 67. Remarkably, as in murine Ngb, the heme binds to the globin in two orientations in Chironomus Hb (42, 43).

In the absence of His64 as the sixth ligand, one would normally expect a pentacoordinate high spin iron in the ferrous deoxy form, with a red-shifted Soret band peaking at 430–440 nm. Therefore, we assign the shoulder at 436 nm in the spectra in Fig. 3b to the five-coordinate species. The predominant species, however, peaks at 422 nm, which is characteristic of hexacoordinate low spin heme. Previously, we had shown that a deoxy Mb species, with a photodissociable water (or OH–) ligand at the sixth coordination, can be generated by photo reduction of aquomet Mb at cryogenic temperature (44). In Ngb, the fast geminate rebinding of CO to the pentacoordinate species at cryogenic temperature (27) and the fast bimolecular CO binding at ambient temperature (Fig. 6) both indicate that the heme iron is extremely reactive toward ligands, as is also expected from the iron out-of-plane displacement toward the distal side by 0.2 Å (14). Therefore, it appears reasonable that Ngb H64L has already substantial affinity for water/OH– even at room temperature. Further support comes from the cryospectroscopy data. Upon cooling, the equilibrium shifts from the pentacoordinate to the hexacoordinate form (Fig. 4c), as expected for a bimolecular reaction for entropic reasons. Moreover, the photo-induced spectral changes further confirm the presence of a small ligand at the sixth coordination. We have noticed that the band at 418 nm (in cryosolvent) grows at the expense of the red shoulder as the pH is raised from 6–10 (data not shown), which may suggest that the photolyzable ligand is an OH– ion. The origin of the third peak at 428 nm is not yet clear; it could be due to binding of water as in photo-reduced met Mb (44).

In both ferric and deoxy Ngb H64L, there is another change in ligation state at alkaline pH (Fig. 3, b and c). Above pH 9, the spectra strongly resemble those of native deoxy Ngb, with sharp {alpha} and {beta} bands typical of two nitrogenous ligands coordinated axially to the heme iron. We associate these changes with ligation of the Lys67 amino group to the heme iron after deprotonation, which happens with a pK of 9.9 (Fig. 2b). This process was also seen in Raman spectra of human Ngb by Uno et al. (28). Consistent with this assignment, drastic changes at high pH are absent in the spectra of ferrous H64L/K67L Ngb (Fig. 3, e and f). Coordination of the heme iron by non-histidine residues in the distal pocket has also been reported for Synechocystis hemoglobin (15, 45). Anyhow, the distal histidine is clearly the primary endogenous ligand in Ngb and a variety of other hemoglobins (46–48).

In ferric H64L/K67L Ngb, the alkaline transition occurs with a pK of 8.8, 1.5 pH units higher than in H64L Ngb. The positively charged Lys67 side chain may assist in binding the OH– ion; therefore, the pK shift upon replacement of Lys67 by leucine occurs likely because of the lacking electrostatic stabilization by a positively charged group.

Much to our surprise, another protonation transition was found at even higher pH in ferric H64L/K67L from the analysis of the Soret shift. This rather sharp transition (Fig. 2c) cannot be described by a simple Henderson-Hasselbalch relation involving a single proton (n = 1 in Equation 1); it represents a cooperative transition involving three or four protonating groups. We also noticed that, at pH > 8.5, the absorption spectra changed very slowly with time and took hours to reach equilibrium. These features are characteristic of partial denaturation and subsequent refolding into a nonnative conformation. A similar phenomenon was also observed with the double mutant H46L/Q43L of Synechocystis hemoglobin (45). There, Hvitved et al. (45) suggested that the double mutation created a highly unstable distal pocket so that other residues can bind to the sixth coordination of the heme iron. A likely ligand for hexacoordination in Ngb H64L/K67L at pH > 8.5 is Arg66 (Fig. 1).

Ligand Binding and Conformational Changes—Flash photolysis kinetics yield information on dynamic processes after photodissociation and intermediate states. At ambient temperature, one usually observes a fast geminate phase on nanosecond to microsecond time scales (in aqueous solvent). For example, geminate rebinding in MbCO and MbO2 extends out to almost 1 µs, indicating that ligands spend up to 1 µs within the protein after bond cleavage, during which they either recombine or escape into the solvent (49). In NgbCO, a pronounced geminate phase extending out to 100 ns was visible at 275 K (27). At higher temperatures, this process becomes faster than the ~30-ns time resolution of our instrument (Figs. 5 and 6). The fast geminate rebinding reflects the low enthalpy barriers at the heme iron (27). The fact that solvent rebinding is nevertheless the dominant kinetic process near physiological temperature indicates that escape of CO from the protein must be very efficient.

In samples equilibrated with 0.05 bar CO, there is essentially no ligand binding between 30 ns and 100 µs (Figs. 5, 6, 7). The deligated species is characterized by a broad spectrum approximately 432 nm. By contrast, the five-coordinate species in NgbCO H64L and H64L/K67L are both centered on 436 nm. This considerable shift to the blue in the wt NgbCO photoproduct may indicate partial occupation of the sixth coordination by water after CO escape.

The first step in the kinetics with apparent rate coefficient {lambda}1 arises from the binding of the exogenous ligand CO from the solvent with rate coefficient {lambda}CO, plus binding of the endogenous ligand (predominantly His64 at physiological pH) with rate coefficient {lambda}His. Under our experimental conditions, with [CO] >> [Ngb], {lambda}CO is a pseudo-first order rate coefficient proportional to [CO]. In a simple kinetic scheme, in which CO and His64 compete for the sixth coordination with unique rate coefficients, the apparent rate coefficient, {lambda}1, and the amplitude of the second step, N2, which is proportional to the yield of His64 binding, are given by the following equations.

(Eq. 3)

(Eq. 4)
Here, C is a proportionality constant. At 1 bar CO, N2 is small because {lambda}CO >> {lambda}His, and {lambda}1 {approx} {lambda}CO. CO association is more than 2 orders of magnitude faster than in MbCO (Ref. 49 and Table I), which again reflects the high reactivity of the heme iron in Ngb. At 0.05 bar CO, {lambda}1 is much smaller because of the decreased [CO], proving that CO binding is indeed a bimolecular reaction. Moreover, both {lambda}His and {lambda}CO are of the same order of magnitude, and there is a markedly increased fraction of endogenous ligands, N2 (Figs. 6 and 7). The second plateau in the kinetics persists up to the lifetime of the His64-iron bond. After thermal dissociation, the His64 ligand is finally replaced by the more strongly bound CO, {lambda}2 {approx} koff (His64). The shortening of the plateau at pH 4 (Fig. 7, a and b) hence signals destabilization of the His64-iron bond under acidic conditions.

The pH dependence of {lambda}1 ({approx}{lambda}CO at 1 bar CO) is most interesting. Fig. 8 clearly shows that {lambda}CO increases by more than 4-fold with pH according to Henderson-Hasselbalch relations with pK values of 6.0 and 6.3 for wt and K67L Ngb, respectively. The comparison with the H64L mutants in Fig. 8 shows that {lambda}CO is much faster and pH-independent if the bulky His64 is replaced by the apolar leucine. Clearly, His64 must be responsible for the pH dependence of {lambda}CO, and it hinders ligand access to the binding site. Interestingly, the Soret spectral shifts revealed much lower pK values of 4.4 and 5.6 for wt and K67L Ngb for His64. These differences indicate a markedly different environment of the His64 imidazole in the equilibrium CO-bound form and the five-coordinate deoxy photoproduct species, in which the distal heme pocket appears quite solvent accessible. In MbCO, the low-pH, protonated species binds CO faster because removal of His64 from the distal heme pocket upon protonation allows much easier ligand access to the active site (35). NgbCO shows the opposite pH dependence of {lambda}CO. The slow CO binding to the low-pH Ngb photoproduct species most likely arises from the better solvent accessibility of the large distal pocket. Water may replace His64 and cause even more hindrance for exogenous ligands to access the heme iron than with His64 in the charge-neutral form inside the distal pocket.

Why then does the presence of water in the distal heme pocket of H64L Ngb not cause a substantial decrease in the CO association rate? The H64L Ngb deoxy spectra show approximately the same amount of five- and six-coordinate heme at room temperature. The on and off rates for water are thus about equal, and the on rate for water will be much faster than for CO because [CO] = 1 mM, and [H2O] = 55 M. Both the on and off rates are thus expected to be fast compared with CO binding, and the CO on rate of H64L will be lowered by only a factor of ~2.

An even more complicated effect of protonation is visible in the pH dependence of the second kinetic step, N2 (Fig. 9). According to Equation 4, this quantity depends on both {lambda}CO and {lambda}His (or, more generally, the rate of any endogenous ligand); it reflects the competition between the exogenous and endogenous ligand. The displacement between the curves at 0.05 and 1 bar CO again shows the bimolecular character of CO binding. However, there is also a pronounced change in N2 with pH. For native Ngb, N2 has small values at very low (pH 4) and intermediate pH (8–9) and maxima approximately pH 6 and toward very high pH values.

The steep increase of N2 above pH 9 in wt NgbCO is absent in the K67L mutant. This suggests that Lys67 acts as a second endogenous ligand at high pH when it becomes deprotonated. The observed increase of N2 by almost 1 order of magnitude shows that Lys67 is the predominant ligand at high pH. This interpretation is consistent with the spectroscopic data on the H64L mutant. There, we had assigned a protonation with pK 9.9 to Lys67. Thus, we conclude that, at alkaline pH, the lysine side chain competes with the exogenous ligand and the His64 imidazole for the sixth coordination in the native protein. The second step appearing in the ligand association kinetics of H64L NgbCO at pH > 9 (Fig. 7c) supports this line of argument. Note that this step is rather short (extending the kinetic decay by a factor of ~3 at pH 10.6). Whereas the association rate of Lys67 is faster than the one of His64 (in wt NgbCO) at high pH, the Lys67-iron bond is so unstable that it persists only up to 1 ms (in H64L NgbCO) before CO replaces the bound Lys67. Moreover, {lambda}1 of H64L becomes faster at high pH, which reflects better access of CO to the active site, most likely because the salt bridge between Lys67 and the heme propionate no longer exists in the neutral form of the Lys67 side chain.

His64 is responsible for the N2 variations below pH 8. Two protons are attached to the imidazole side chain at low pH, whereas the neutral state has two tautomeric forms: one with a proton at N{epsilon}2 and another one with the proton on N{delta}1. The first tautomer is known to be more stable in aqueous solution. However, only the second tautomer can bind to the heme iron by means of the lone pair on N{epsilon}2. At low pH, the distal histidine is most of the time in the charged conformation outside the distal heme pocket so that it cannot bind to the heme iron. As a consequence, the rate coefficient {lambda}His and the yield N2 are small. At pH 8, the distal histidine is predominantly neutral and in the distal pocket in its more stable N{epsilon}2 tautomeric state, which cannot bind to the heme iron. For both wt and K67L NgbCO, the maximum yield of His64-complexed heme occurs at the titration midpoint (Figs. 8 and 9), which is characterized by the largest number of transitions between the protonated and unprotonated forms. We believe that, under the nonequilibrium conditions of the ligand binding experiments, this may lead to the maximum probability of finding the N{delta}1 species in the heme pocket and thus the highest yield of His64-coordinated heme.

Conclusions—Even though the physiological role of Ngb still awaits further clarification, the binding of small, gaseous ligands will likely be a key event in its function. In this work, we have used both steady-state and time-resolved visible spectroscopy to examine the role of distal pocket residues and their protonation states on ligand binding to ferric and ferrous Ngb. By using mutants, we were able to assess the detailed roles of His64 and Lys67. His64 clearly provides significant hindrance to access of exogenous ligands to the active site. Lys67 sequesters the distal pocket from the solvent environment. A variety of different heme complexes exist in the ferric and ferrous states, and His64 and Lys67 can both compete as endogenous ligands. The substantial changes in pK observed for the His64 imidazole side chain under different conditions indicate a highly flexible distal pocket, with conformations varying greatly with respect to solvent accessibility. Most remarkable is the enormous reactivity of the heme iron toward ligands in Ngb, as reflected in the very large association rates for CO and the ability of the H64L mutant to bind H2O or OH– at the sixth coordination. The major structural determinant of the exceptional reactivity is likely the out-of-plane positioning of the iron toward the distal side (14). A large fraction of bimolecular rebinding indicates that there are highly efficient ligand escape routes that prevent geminate recombination. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH and may thus be relevant to the function of Ngb.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant Ni-291/3 and by the Fonds der Chemischen Industrie. 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. Back

To whom correspondence should be addressed: Dept. of Physics, University of Illinois at Urbana-Champaign, 1110 W. Green, Urbana, IL 61801. E-mail: uli{at}uiuc.edu.

1 The abbreviations used are: Hb, hemoglobin; Ngb, neuroglobin; Mb, myoglobin; wt, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Drs. T. Burmester and T. Hankeln (University of Mainz, Germany) for kindly providing the murine Ngb expression system, Uwe Theilen for constructing and producing the neuroglobin mutants used in this study, and Florian Junginger for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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