FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

A comparative study on axial coordination and ligand binding in ferric mini myoglobin and horse heart myoglobin

The absorption and resonance Raman spectra and the azide binding kinetics of ferric horse heart myoglobin (Mb) and mini myoglobin (a chemically truncated form of horse heart Mb containing residues 32-139) have been compared. The steady-state spectra show that an additional six-coordinated low-spin form (not present in entire horse heart Mb, which is purely six-coordinated high spin) predominates in mini Mb. The distal histidine is possibly the sixth ligand in this species. The presence of two species corresponds to a kinetic biphasicity for mini Mb that is not observed for horse heart Mb. Azide binds to horse heart Mb much more slowly than to sperm whale Mb. This difference may result from a sterically hindered distal pocket in horse heart Mb. In both cases, the rate constants level off at high azide concentrations, implying the existence of a rate-limiting step (likely referable to the dissociation of the axial sixth ligand). The faster rate constant of mini Mb is similar to that of sperm whale Mb, whereas the slower one is similar to that of entire horse heart Mb.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

Kinetic studies of spin interconversion in ferric mixed-spin derivatives of myoglobin

Kinetic studies of spin interconversion in various derivatives of metmyoglobin such as the fluoride, aquo, hydroxide, azide, imidazole, and cyanide were performed by the coaxial-cable temperature-jump method. For all these derivatives, except fluoride and aquomyoglobin, a single relaxation was observed around 3 μsec. The rate constants and activation parameters for the spin interconversion were estimated and are discussed in comparison with those reported for the reaction of synthetic iron complex. Other hemoproteins such as cytochrome c and human hemoglobin were also examined, and the results were compared with those for myoglobin. The effect of buffer solution is also discussed.     

Axial coordination of ferric Aplysia myoglobin

Resonance Raman spectra of ferric Aplysia myoglobin in the ligand-free and the azide-bound forms have been studied over a wide pH range to determine the coordination states of the heme iron atom. In the hydroxide form at high pH (approximately 9) the iron is six-coordinate and is in a high/low spin equilibrium. As the pH is lowered below the acid/alkaline transition (pKa = 7.5), the heme becomes five-coordinate. When the pH is lowered even further no other changes in the resonance Raman spectrum are detected; thus, the heme remains five-coordinate down to pH 4, the lowest value studied. For ferric azide-bound Aplysia myoglobin, the iron is six-coordinate in a high/low spin equilibrium at all pH values (4.8-9). These data indicate (i) that the unusual reactivity toward azide previously observed at neutral pH is indeed related to the absence of a coordinated water molecule, and (ii) that causes other than the heme coordination are responsible for the spectral differences and the ligand-binding kinetics differences observed below pH 6.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Resonance Raman spectroscopic studies of hydroperoxo derivatives of cobalt-substituted myoglobin

Recent progress in generating and stabilizing reactive heme protein enzymatic intermediates by cryoradiolytic reduction has prompted application of a range of spectroscopic approaches to effectively interrogate these species. The impressive potential of resonance Raman spectroscopy for characterizing such samples has been recently demonstrated in a number of studies of peroxo- and hydroperoxo-intermediates. While it is anticipated that this approach can be productively applied to the wide range of heme proteins whose reaction cycles naturally involve these peroxo- and hydroperoxo-intermediates, one limitation that sometimes arises is the lack of enhancement of the key intraligand ν(O-O) stretching mode in the native systems. The present work was undertaken to explore the utility of cobalt substitution to enhance both the ν(Co-O) and ν(O-O) modes of the CoOOH fragments of hydroperoxo forms of heme proteins bearing a trans-axial histidine linkage. Thus, having recently completed RR studies of hydroperoxo myoglobin, attention is now turned to its cobalt-substituted analogue. Spectra are acquired for samples prepared with ¹⁶O₂ and ¹⁸O₂ to reveal the ν(M-O) and ν(O-O) modes, the latter indeed being observed only for the cobalt-substituted proteins. In addition, spectra of samples prepared in deuterated solvents were also acquired, providing definitive evidence for the presence of the hydroperoxo-species.     

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.     

Resonance Raman studies of CO and O2 binding to elephant myoglobin (distal His(E7)----Gln)

Carbon monoxide and dioxygen were employed as resonance Raman-visible ligands for probing the nature of the heme-binding site in elephant myoglobin, which has glutamine in the distal position (E7) instead of the usual histidine. The distal histidine (E7) residue has been thought to be responsible for weakening carbon monoxide binding to hemoproteins. It is of interest to see how the His(E7)----Gln replacement affects such parameters as nu(Fe-N epsilon), nu(Fe-CO), delta(Fe-C-O), nu(C-O), delta(Fe-O-O), and nu(O-O) vibrational frequencies and relative intensities. Elephant myoglobin has a CO affinity approximately 6 times higher than that for human/sperm whale myoglobin (Mb). If this enhanced affinity were solely due to the removal of some of the steric hindrance that normally tilts the CO off the heme axis, one would expect the nu(Fe-CO) frequency to decrease and the nu(C-O) frequency to increase relative to the corresponding values in sperm whale Mb. However, the opposite was found. In addition, strong enhancement of the Fe-C-O bending mode was observed. These results suggest that the Fe-C-O linkage remains distorted. In elephant Mb, new interactions resulting from the conformational change accompanying ligand binding may be responsible for the increased CO binding. Similar spectra were obtained for elephant and sperm whale oxymyoglobin. This suggests that the interactions of bound O2 are not markedly affected by the glutamine replacement.     

Resonance Raman enhancement of phenyl ring vibrational modes in phenyl iron complex of myoglobin.

Resonance Raman spectra are reported for the organometallic phenyl-FeIII complexes of horse heart myoglobin. We observed the resonance enhancement of the ring vibrational modes of the bound phenyl group. They were identified at 642, 996, 1,009, and 1,048 cm-1, which shift to 619, 961, 972, and 1,030 cm-1, respectively, upon phenyl 13C substitution. The lines at 642 and 996 cm-1 are assigned, respectively, as in-plane phenyl ring deformation mode (derived from benzene vibration No. 6a at 606 cm-1) and out-of-plane CH deformation (derived from benzene vibration No. 5 at 995 cm-1). The frequencies of the ring "breathing" modes at 1,009 and 1,048 cm-1 are higher than the corresponding ones in phenylalanine (at 1,004 and 1,033 cm-1) and benzene (at 992 and 1,010 cm-1), indicating that the ring C--C bonds are strengthened (or shortened) when coordinated to the heme iron. The excitation profiles of these phenyl ring modes and a porphyrin ring vibrational mode at 674 cm-1 exhibit peaks near its Soret absorption maximum at 431 nm. This appears to indicate that these phenyl ring modes may be enhanced via resonance with the Soret pi-pi transition. The FeIII--C bond stretching vibration has not been detected with excitation wavelengths in the 406.7-457.9-nm region.     

Theoretical characterization of carbon monoxide vibrational spectrum in sperm whale myoglobin distal pocket

In this article we use the perturbed matrix method and an extended molecular dynamics sampling of the carbon monoxide (CO) in the myoglobin distal pocket to characterize the CO vibrational spectrum and hence to relate its spectroscopic features with the atomic-molecular behavior. Results show the accuracy of the method employed and confirm the assignment of the spectroscopic B1 and B2 states proposed by Lim et al.     

Resonance Raman investigation of ferric iron in horseradish peroxidase and its aromatic donor complexes at room and low temperatures

Resonance Raman (RR) spectra of the acidic form of FeIII horseradish peroxidase (HRP) were obtained at room and low temperatures using B- and Q-band excitation. At 296 K, HRP exhibits two sets of porphyrin skeletal stretching frequencies which are attributed to a thermal mixture of 5- and 6-coordinate high-spin FeIII states. When the temperature is lowered, the observed bands shift to higher frequencies, and these are assigned to intermediate- and low-spin states. Addition of 40% glycerol has no effect on the spectra at 296 K, but at 20 K, all four frequency sets are observed corresponding to the two forms observed at room and low temperature in the absence of glycerol. The 296 K RR spectrum of the HRP-hydroquinone complex is similar to that of free HRP, but conversion to the intermediate- and low-spin states is complete at a higher temperature than in the free enzyme. Addition of benzohydroxamic acid (BHA) to HRP shifts the RR frequencies to those corresponding to a 6-coordinate high-spin species at both room and low temperature. Two upsilon (C = C) stretching modes are observed for HRP and its donor complexes, indicating that the vinyl groups are inequivalent. On BHA binding, one of the vinyl modes and upsilon 37 (Eu) are enhanced, suggesting symmetry lowering of the heme site.     

Resonance Raman studies of ETE dehydrogenase (an iron sulfur flavoprotein)

ETF Dehydrogenase is an iron sulfur flavoprotein responsible for the transfer of electrons between electron transfer flavoprotein (ETF) and CoQ of the electron transport chain. We have determined the resonance Raman spectrum of this enzyme observing in the process at least seven of thirteen flavin bands in the 1100cm-1-1600 cm-1 region of the Raman spectrum. The positions of three of these bands, II, IX, and X (see Figure I and Table I for band numbering system) in ETF dehydrogenase is very similar to their positions in aqueous solution of flavins in which water is hydrogen bonded to N-1, N-5, C=0(2), C=0(4), and N-H(3) of flavin. Conversely the positions of the flavin Raman bands are considerably shifted from those of flavin in nonhydrogen bonding solvent. The positions of bands II, IX, and X are nearly identical to those in the flavoprotein glutathione reductase; x-ray structural investigations on this enzyme indicate that there is extensive hydrogen bonding between FAD and protein in this molecule. A previous study in our laboratory has demonstrated that metal complexation at N-5 and C=0(4) with either Ru or Ag produces large shifts in the positions of Raman bands II, VI, IX, and X. None of these shifts are observed in ETF dehydrogenase indicating that there is no direct inner sphere coordination of Fe to flavin. In addition to the Raman bands of flavin observed in our spectrum, we also observe one band that is in the Fe-S stretching region observed for a variety of Fe-S proteins. This band is located at 331 cm-1. The frequency of the band corresponds to the 335 cm-1 band associated with the strongest Fe-S stretching mode in the 4Fe-4S protein ferrodoxin from C. pasterianum. The observed frequency is quite different from that of the 3Fe-3S proteins such as ferrodoxin(II) from D. gigas. Finally, ETF dehydrogenase shows no loss of activity or visual evidence of photodegradation in the laser beam as most other FeS proteins do.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

Resonance Raman studies of lactoperoxidase

Resonance Raman (RR) spectra obtained at three excitation wavelengths are reported for various ferric, ferrous, and ferryl derivatives of bovine lactoperoxidase. The RR spectra of the ferric derivatives show the full complement of the vinyl stretching and scissor modes indicating that the two vinyls in the protoporphyrin IX prosthetic group are present in unmodified forms. The cysteine thiol complex exhibits a RR spectrum identical to that of the native enzyme, an observation which strongly suggests a nonheme binding site for the thiol substrates. The different ferrous complexes of lactoperoxidase which result from heme reduction at slightly alkaline and acidic pH gave identical low-frequency RR spectra. Differences are observed, however, in the high-frequency region. Reduction in the presence of cyanide, however, yields two time-resolved complexes. Changes in the ligand field during the conversion to the final form of the cyanoferrous complex are proposed based on the changes observed in the low-frequency vibrational spectrum. Comparisons are made between the low-frequency RR spectra of the limiting form of the cyanoferrous and the nitric oxide lactoperoxidase complexes. The similarity between the RR spectra of these two complexes in the 150-500 cm-1 region supports the assignment of structures for these complexes where the six-coordinate heme iron is displaced from the heme plane and away from the proximal histidine ligand.     

Transition between iron spin states in myoglobin: Roentgen absorption

The spin transition of Fe in the active center of a myoglobin molecule, stimulated by a temperature variation, was studied by the theoretical multiple scattering analysis of experimental X-ray absorption data. The spin transition was followed by the movement of the Fe ion out of the plane of the heme without substantial changes in the Fe-N distance. It was shown that the X-ray absorption fine structure above the Fe K-edge is sensitive to both local geometry changes near the Fe ion in the active site of the protein and the spin state of the Fe ion itself. The change in the symmetry of Fe coordination lead to modifications of the spectrum shape in the entire interval up to 40 eV above the main edge. It was found that the spin state effects mostly the rising edge, and at energies above 15 eV becomes negligibly small.     

Time-resolved hole-burning study on myoglobin: fluctuation of restricted water within distal pocket

We have studied the equilibrium fluctuation dynamics of Zn-substituted myoglobin and its His64-->Leu (H64L) mutant in the pH range from 5 to 9 by using time-resolved transient-hole-burning (TRTHB) spectroscopy. In the H64L mutant, we have observed a largely reduced width of the absorption spectrum and only a slight temporal shift of the hole-burning spectrum. These observations both reflect the suppressed conformational fluctuation in the mutant. On the other hand, the pH-dependent change in the absorption spectrum could not be solely explained by the change in the protonation state of His64 induced by the pH change. These results suggest that although the fluctuation dynamics observed by the TRTHB experiment of the native sample mainly reflects the conformational motion around His64, the interconversion process of His64 between its protonated and unprotonated states has a minor contribution. Instead, we have proposed a tentative interpretation that the motion of the water molecule around His64 is the main source of the observed dynamics in the TRTHB technique.     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.