Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.     

Electronic and stereochemical characterizations of the photoinduced intermediates of nitrosyl complexes of metal (S = 5/2)-substituted hemoproteins trapped at low temperature

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.     

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.     

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.     

Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.     

Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low-spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein, the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87, suggested by the observation of a H/D sensitive resonance Raman line at 502 cm(-)(1) that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.     

Ligand binding to heme proteins: III. FTIR studies of His-E7 and Val-E11 mutants of carbonmonoxymyoglobin.

Fouier-transform infrared (FTIR) difference spectra of several His-E7 and Val-E11 mutants of sperm whale carbonmonoxymyoglobin were obtained by photodissociation at cryogenic temperatures. The IR absorption of the CO ligand shows characteristic features for each of the mutants, both in the ligand-bound (A) state and in the photodissociated (B) state. For most of the mutants, a single A substate band is observed, which points to the crucial role of the His-E7 residue in determining the A substrate spectrum of the bound CO in the native structure. The fact that some of the mutants show more than one stretch band of the bound CO indicates that the appearance of multiple A substates is not exclusively connected to the presence of His-E7. In all but one mutant, multiple stretch bands of the CO in the photodissociated state are observed; these B substates are thought to arise from discrete positions and/or orientations of the photodissociated ligand in the heme pocket. The red shifts of the B bands with respect to the free-gas frequency indicate weak binding in the heme pocket. The observation of similar red shifts in microperoxidase (MP-8), where there is no residue on the distal side, suggests that the photodissociated ligand is still associated with the heme iron. Photoselection experiments were performed to determine the orientation of the bound ligand with respect to the heme normal by photolyzing small fractions of the sample with linearly polarized light at 540 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

FTIR and resonance Raman studies of nitric oxide binding to H93G cavity mutants of myoglobin.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a five- or six-coordinate Fe-NO complex. The H93G mutation eliminates the covalent attachment between the protein and the proximal ligand, allowing NO to bind H93G possibly from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side. The question of whether NO binds on the distal or proximal side was addressed by FTIR spectroscopy of the N-O vibrational frequency nuN(-O) for a set of Mb mutants that perturb the electrostatic environment of the heme pocket. Vibrational spectra of five- and six-coordinate MbNO complexes indicate that nu(N-O) shifts (by as much as 26 cm(-1)) to higher energies for the distal mutants H64V and H64V/H93G relative to the energies of wild-type and H93G MbNO, while nu(N-O) is not affected by the proximal side mutation S92A/H93G. This result suggests that NO binds on the distal side of heme in the five- and six-coordinate MbNO complexes of H93G. Additionally, values of the Fe-NO vibrational frequency nu(Fe-NO) as measured by resonance Raman spectroscopy are reported for the distal and proximal double mutants of H93G. These results suggest that nu(Fe-NO) is not very sensitive to mutations that perturb the electrostatic environment of the heme pocket, leading to the observation that nu(N-O) and nu(Fe-NO) are not quantitatively correlated for the MbNO complexes presented here. Furthermore, nu(N-O) and nu(Fe-NO) do not correlate well with equilibrium constants for imidazole binding to the five-coordinate MbNO complexes of the H93G double mutants. The data presented here do not appear to support the presence of pi-back-bonding or an inverse trans effect of NO binding in Mb mutants that alter the electrostatic environment of the heme pocket.     

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.     

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.     

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin

Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of results using Soret band excitation with previous Q-state excitation studies demonstrates that the ligand dissociates with a similar kinetic energy in both cases, suggesting fast intramolecular energy redistribution before dissociation.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hexaheme nitrite reductase from Desulfovibrio desulfuricans. M?ssbauer and EPR characterization of the heme groups

M?ssbauer and EPR spectroscopy were used to characterize the heme prosthetic groups of the nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774), which is a membrane-bound multiheme cytochrome capable of catalyzing the 6-electron reduction of nitrite to ammonia. At pH 7.6, the as-isolated enzyme exhibited a complex EPR spectrum consisting of a low-spin ferric heme signal at g = 2.96, 2.28, and 1.50 plus several broad resonances indicative of spin-spin interactions among the heme groups. EPR redox titration studies revealed yet another low-spin ferric heme signal at g = 3.2 and 2.14 (the third g value was undetected) and the presence of a high-spin ferric heme. M?ssbauer measurements demonstrated further that this enzyme contained six distinct heme groups: one high-spin (S = 5/2) and five low-spin (S = 1/2) ferric hemes. Characteristic hyperfine parameters for all six hemes were obtained through a detailed analysis of the M?ssbauer spectra. D. desulfuricans nitrite reductase can be reduced by chemical reductants, such as dithionite or reduced methyl viologen, or by hydrogenase under hydrogen atmosphere. Addition of nitrite to the fully reduced enzyme reoxidized all five low-spin hemes to their ferric states. The high-spin heme, however, was found to complex NO, suggesting that the high-spin heme could be the substrate binding site and that NO could be an intermediate present in an enzyme-bound form.     

Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding

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.     

Distal Val346Ile mutation in inducible NO synthase promotes substrate-dependent NO confinement

The function of inducible NO synthase (WT iNOS) depends on the release of NO from the ferric heme before the enzyme is reduced. Key parameters controlling ligand dynamics include the distal and proximal heme pocket amino acids, as well as the inner solvent molecules. In this work, we tested how a point mutation in the distal heme side of WT iNOS affected the geminate rebinding of NO by ultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodissociated NO close to the heme compared to WT iNOS, with a main picosecond phase accounting for 78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO release from Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site is filled. These data are rationalized by a steric effect of the Ile methyl group inducing events mediated by the substrate, transmitted via the propionates to the NO and the protein. This model is consistent with the role of the H-bonding network involving the heme, the substrate, and the BH4 cofactor in controlling NO release, with a key role of the heme propionates [Gautier et al. (2006) Nitric Oxide 15, 312]. These data support the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesis compared to WT iNOS determined by single turnover catalysis [Wang et al. (2004) J. Biol. Chem. 279, 19018].     

Resonance Raman studies of iron spin and axial coordination in distal pocket mutants of ferric myoglobin

We have used resonance Raman spectroscopy to study 11 distal pocket mutants and the "wild type" and native ferric sperm whale myoglobin. The characteristic Raman core-size markers v4, v3, v2, and v10 are utilized to assign the spin and coordination state of each sample. It is demonstrated that replacements of the distal and proximal histidines can discriminate against H2O as a sixth ligand and favor a pentacoordinate Fe3+ atom. Soret absorption band blueshifts are correlated with the pentacoordinate heme environment. One E7 replacement (Arg) leads to an iron spin state change and produces a low spin species. The Glu and Ala mutations at position E11 leave the protein's spin and coordination unaltered. A laser-induced photoreduction effect is observed in all pentacoordinate mutants and seems to be correlated with the loss of the heme-bound water molecule.     

The effect of ligand dynamics on heme electronic transition band III in myoglobin.

Band III is a near-infrared electronic transition at ~13,000 cm(-1) in heme proteins that has been studied extensively as a marker of protein conformational relaxation after photodissociation of the heme-bound ligand. To examine the influence of the heme pocket structure and ligand dynamics on band III, we have studied carbon monoxide recombination in a variety of myoglobin mutants after photolysis at 3 K using Fourier transform infrared temperature-derivative spectroscopy with monitoring in three spectral ranges, (1) band III, the mid-infrared region of (2) the heme-bound CO, and (3) the photodissociated CO. Here we present data on mutant myoglobins V68F and L29W, which both exhibit pronounced ligand movements at low temperature. From spectral and kinetic analyses in the mid-infrared, a small number of photoproduct populations can be distinguished, differing in their distal heme pocket conformations and/or CO locations. We have decomposed band III into its individual photoproduct contributions. Each photoproduct state exhibits a different "kinetic hole-burning" (KHB) effect, a coupling of the activation enthalpy for rebinding to the position of band III. The analysis reveals that the heme pocket structure and the photodissociated CO markedly affect the band III transition. A strong kinetic hole-burning effect results only when the CO ligand resides in the docking site on top of the heme group. Migration of CO away from the heme group leads to an overall blue shift of band III. Consequently, band III can be used as a sensitive tool to study ligand dynamics after photodissociation in heme proteins.     

Assignment of proton resonances in the NMR spectrum of carbonmonoxy hemoglobin beta subunit tetramers

Isolated beta chains from human adult hemoglobin at millimolar concentration are mainly associated to form beta 4 tetramers. We were able to obtain relevant two-dimensional proton nuclear magnetic resonance (NMR) spectra of such supermolecular complexes (Mr approximately 66,000) in the carboxylated state. Analysis of the spectra enabled us to assign the major part of the proton resonances corresponding to the heme substituents. We also report assignments of proton resonances originating from 12 amino acid side chains mainly situated in the heme pocket. These results provide a basis for a comparative analysis of the tertiary heme structure in isolated beta(CO) chains in solution and in beta(CO) subunits of hemoglobin crystals. The two structures are generally similar. A significantly different position, closer to the heme center, is predicted by the NMR for Leu-141 (H19) in isolated beta chains. Comparison of the assigned resonances of conserved amino acids in alpha chains, beta chains and sperm whale myoglobin indicates a close similarity of the tertiary heme pocket structure in the three homologous proteins. Significant differences were noted on the distal heme side, at the position of Val-E11, and on Leu-H19 and Phe-G5 position on the proximal side.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.