Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis

The reaction with carbon monoxide of the cooperative dimeric hemoglobin from Scapharca inaequivalvis has been examined by flash photolysis. In the nanosecond time range, geminate rebinding of 5% of dissociated CO occurs with a rate constant of 1.4 x 10(7) s-1. There is a change in absorbance of deoxyhemoglobin following photolysis at a rate of 1.2 x 10(6) s-1, consistent with a shift in the position of the Soret band to longer wavelengths. The amplitude of the change is proportional to the population of deoxydimer. In much of the Soret region this change is greater than the absorbance excursion associated with geminate recombination. There is at least one other slower change associated with the singly liganded species. Geminate rebinding of NO has components of 50, 8, and 0.035 ns-1, accounting for 75%, 25%, and less than 1% of the total reaction observed after a 35-ps photolysis flash. Simulation of diffusion of NO by molecular dynamics shows the ligands moving from the heme pocket to a subsidiary space between the edge of the heme and the surface of the protein.     

The effects of behavioral tasks on the in vitro phosphorylation of intermediate filament subunits of rat hippocampus are mediated by CaMKII and PKA

In the homodimeric hemoglobin from Scapharca, HbI, functional communication between the two heme groups is based on their direct structural linkage across the subunit interface through the heme propionates. The heme-protein interactions have been altered in deutero- and meso-HbI by substituting the vinyl groups at positions 2 and 4 of protoheme with hydrogen and ethyl groups, respectively. In meso-HbI the introduction of the ethyl groups in the heme pocket induces significant alterations in the conformation of the heme peripheral substituents, including the propionates, and in the structure of bound CO, as revealed by the resonance Raman spectra. The functional counterpart of these structural changes is the loss of cooperativity in carbon monoxide binding and in the rate of oxygen dissociation. Oxygen pulse and flash photolysis experiments indicate that meso-HbI is locked in the liganded conformation. It is postulated that the ethyl groups, which occupy a larger volume than vinyl ones, impair the ligand-linked movement of the heme relative to its pocket and in turn the expression of cooperativity. In deutero-HbI structural alterations have not been monitored. Functionally, cooperativity in the CO binding kinetics is increased as if hydrogen atoms at positions 2 and 4 permitted more marked movements of the heme than in the native protein.     

A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata)

The architecture of the distal heme pocket in hemoglobins and myoglobins can play an important role in controlling ligand binding dynamics. The size and polarity of the residues occupying the distal pocket may contribute steric and dielectric effects. In vertebrate systems, the distal pocket typically contains a "distal" histidine at position E7 and a leucine at position B10. There are several invertebrate organisms that have hemoglobins or myoglobins that display a pattern in which residues E7 and B10 are a glutamine and tyrosine, respectively. These proteins often have very high oxygen affinities stemming from very slow ligand off rates. In this study, two such hemoglobins, one from the nematode Ascaris suum and the other from the sulfide-fixing clam Lucina pectinata, are compared with respect to conformational and functional properties. Ultraviolet resonance Raman spectroscopy and visible resonance Raman spectroscopy are used to probe, respectively, the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal heme pocket interactions. Fourier transform infrared absorption spectroscopy is used to probe the dielectric properties of the distal heme pocket through the stretching frequency of carbon monoxide bound to the heme. Functionality is probed through the geminate rebinding of both CO and O2. The findings reveal two very different patterns indicative of two different mechanisms for achieving low oxygen off rates. In Hb Ascaris, a hydrogen bonding network that includes the E7 Gln, B10 Tyr, and oxygen bound to the heme results in a tight cage for the oxygen. Dissociation of the O2 requires a large amplitude conformational fluctuation that results both in a spontaneous dissociation of the oxygen through the loss of hydrogen bond stabilization and in an enhanced probability for ligand escape though the transient disruption and opening of the tight distal cage. In the case of the Hb from Lucina, there is no evidence for a tight cage. Instead the data support a model in which the hydrogen bonding network is far more tenuous and the equilibrium state of distal pocket is far more open and accessible than is the case in Ascaris. The results explain why Hb Ascaris has one of the highest oxygen affinities known (P50 approximately 10(-)3 Torr) while Hb Lucina II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr) even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates. The roles of water and proximal strain are discussed.     

A carbon monoxide derivative of ruthenium (II) myoglobin probe of heme protein conformation

A synthetic carbon monoxide complex of ruthenium (II) myoglobin has been reconstituted from apomyoglobin and the carbon monoxide of ruthenium (II) mesoporphyrin IX. This synthetic myoglobin complex shows an absorption spectrum with normal Soret and beta bands, and a split alpha band. The alpha-band splitting is not observed in the spectrum of the carbon monoxide derivative of ruthenium (II) mesoporphyrin IX in pyridine, even though the width of the alpha band in pyridine is narrower than in theprotein. The separation between two alpha bands in the spectrum of the protein is reduced from 7.5 to 6 nm in the presence of 2 M NaCl. This observation is interpreted in terms of perturbation of concentrated NaCl on the protein. The separation between the maxima of two alpha bands in the spectrum of the ruthenium (II) myoglobin also becomes smaller with decrease in pH from 7 to 4.5, and this process involves the distal histidine. The alpha band splitting in this protein complex is interpreted in terms of rhombic distortion of the square planar symmetry of the metal porphyrin in the protein.     

Structural interactions between horseradish peroxidase C and the substrate benzhydroxamic acid determined by X-ray crystallography

The three-dimensional structure of recombinant horseradish peroxidase in complex with BHA (benzhydroxamic acid) is the first structure of a peroxidase-substrate complex demonstrating the existence of an aromatic binding pocket. The crystal structure of the peroxidase-substrate complex has been determined to 2.0 A resolution with a crystallographic R-factor of 0.176 (R-free = 0. 192). A well-defined electron density for BHA is observed in the peroxidase active site, with a hydrophobic pocket surrounding the aromatic ring of the substrate. The hydrophobic pocket is provided by residues H42, F68, G69, A140, P141, and F179 and heme C18, C18-methyl, and C20, with the shortest distance (3.7 A) found between heme C18-methyl and BHA C63. Very little structural rearrangement is seen in the heme crevice in response to substrate binding. F68 moves to form a lid on the hydrophobic pocket, and the distal water molecule moves 0.6 A toward the heme iron. The bound BHA molecule forms an extensive hydrogen bonding network with H42, R38, P139, and the distal water molecule 2.6 A above the heme iron. This remarkably good match in hydrogen bond requirements between the catalytic residues of HRPC and BHA makes the extended interaction between BHA and the distal heme crevice of HRPC possible. Indeed, the ability of BHA to bind to peroxidases, which lack a peripheral hydrophobic pocket, suggests that BHA is a general counterpart for the conserved hydrogen bond donors and acceptors of the distal catalytic site. The closest aromatic residue to BHA is F179, which we predict provides an important hydrophobic interaction with more typical peroxidase substrates.     

Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH

Resonance Raman (RR), electronic absorption, and circular dichroism (CD) spectroscopies of the ferric, ferrous, and ferrous-CO forms of horseradish peroxidase (HRP-C) at pH 3.1 are reported. The CD spectra in the UV region show only a small decrease in the alpha-helical content upon pH lowering, whereas dramatic changes are observed in the Soret region. The final form of ferric HRP-C is 5-coordinate high-spin heme whose histidine ligand is replaced by a water ligand with a polar character. The electronic and CD spectra show the presence of an intermediate form with a 6-coordinate heme. Therefore, the cleavage of the proximal Fe-imidazole bond is preceded by the binding of a distal water molecule. For the ferrous form of HRP-C, the pH-dependence of the absorption spectra revealed only the native form in the range pH 5-7 and an unfolded form with a Soret maximum at 383 nm at pH 3.1. An intermediate state, characterized by a Soret maximum at 424 nm, was observed only in a transient way, within a few milliseconds. A metastable and a final species are observed also for the ferrous-CO complex at pH 3.1, as proved by isosbestic points in the electronic absorption spectra. The two forms show different RR nu(Fe-C) and IR nu(CO) modes. The metastable form corresponds to a heme where histidine is replaced by water. The final form is due to the displacement of the water ligand by the proximal histidine. We propose a kinetic model to account for our results at pH 3.1 for the ferric, ferrous, and ferrous-CO forms.     

Orientation of the heme vinyl groups in the hydrogen sulfide-binding hemoglobin I from Lucina pectinata

Hemoglobin I (HbI) from the claim Lucina pectinata is a unique heme protein that binds and transfers hydrogen sulfide (H2S) to a symbiotic bacteria. The metcyano, metaquo, carbon monoxy, oxy, and deoxy complexes of HbI were studies by resonance Raman (RR) spectroscopy, and the metacyano and carbon monoxy complexes were also studied by 1H-NMR. The results indicate a unique orientation of the heme 2-vinyl group relative to other heme proteins. The RR spectra of the HbICO, metHbICN, metHbIH2O, HbIO2 and deoxyHbI heme derivatives show a band at 1621 cm-1 and a shoulder at 1626 cm-1, indicative of an out-of-plane position for one of the vinyls relative to the other one. Spin-lattice relaxation properties of protons in the metHbICN complex also suggest a unique orientation for the heme 2-vinyl group of HbI. The longitudinal relaxation time (T1) for the 2-H alpha, 2-H beta c, and H beta t protons are 120 ms, 115 ms, and 135 ms, respectively. The data from both techniques suggest an out-of-plane and trans-oriented 2-vinyl group, and an in-plane and cis-oriented 4-vinyl group for the low-spin complexes of HbI. These results imply that the electron withdrawing character of the out-of-plane vinyl group contributes to the stability of the heme Fe+3 oxidation state, facilitates the binding of the H2S ligand, and promotes the stability of this ferric H2S complex.     

Solution and crystal structures of a sperm whale myoglobin triple mutant that mimics the sulfide-binding hemoglobin from Lucina pectinata

The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86-89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with approximately 700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.     

Dynamics of cyanide binding to ferrous Scapharca inaequivalvis homodimeric hemoglobin

Flash photolysis experiments have been carried out for the first time on a hemoglobin ferrous cyanide adduct with an 8 ns laser pulse. A 95% nonexponential rebinding process occurs within 2 micros after full photolysis in ferrous cyanide dimeric Scapharca inaequivalvis hemoglobin (HbI), indicating that once photolyzed the cyanide anion is not able to escape from the protein matrix and rebinds to the heme iron. The resonance Raman spectrum of the 10 ns photoproduct is identical to that of the fully relaxed deoxy derivative, indicating that in the ferrous cyanide HbI adduct protein relaxation occurs within 10 ns after photolysis. This behavior is at variance with that of the carbonmonoxy HbI derivative in which very little geminate rebinding is observed and the photoproduct relaxes with a lifetime of 1 micros. The fast relaxation of the cyanide HbI photoproduct can be accounted for by the small perturbation of the heme structure induced by cyanide binding to ferrous HbI. This is consistent with a deoxy-like conformation of the HbI ferrous cyanide adduct and implies that the pathway for relaxation involves only minor local rearrangements of the heme moiety. Photolysis experiments carried out on ferrous cyanide horse myoglobin, which can be saturated only partially, show a qualitatively similar behavior in ligand rebinding, indicating that the geminate process of the cyanide anion is a general phenomenon in hemoproteins.     

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.     

Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.     

Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.     

Oxidative titrations of reduced cytochrome aa3: correlation of midpoint potentials and extinction coefficients observed at three major absorption bands

Anaerobic oxidative titrations of purified cytochrome aa3 were monitored at three wavelengths (444, 604, and 820 nm), in both the absence and the presence of carbon monoxide. Computer simulation of each titration curve was utilized to ascertain the midpoint potentials of the four oxidation-reduction centers of the enzyme. For experiments performed under nitrogen, two components were found to titrate with low potential (heme aL = 220 mV, CuL = 240 mV) and two with high potential (heme ath, cuH = 340 mV), consistent with results obtained previously in reductive titrations. Unequal heme extinction coefficients were observed at 444 nm. Oxidation by either potassium ferricyanide or 1,1'-bis(hydroxymethyl)ferricinium ion showed that the low potential heme component contributed 75% of the absorbance change at 444 nm. At 820 nm, the entire absorbance change could be attributed to a single, low potential copper component. Midpoint potentials calculated for the carbon monoxide complexed enzyme agreed with previously reported values. The copper components retained the values observed under nitrogen, while the titratable heme group gave an apparent midpoint potential of 260 mV. These results enable us to assign absorbance changes at various wavelengths to specific redox components of cytochrome aa3.     

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. The resulting linear dichroism in the CO stretch spectrum yielded angles alpha > 20 degrees between the CO molecular axis and the heme normal for all of the mutants. We conclude that the off-axis position of the CO ligand in the native structure does not arise from steric constraints imposed by the distal histidine. There is no clear correlation between the size of the distal residue and the alpha of the CO ligand.     

Localization of the heme binding region in soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. sGC is activated by nitric oxide (NO) and therefore plays a central role in NO signal transduction. Activation of sGC by NO is believed to be mediated by the interaction between NO and the heme of sGC. Spectroscopic and kinetic studies have shown that the heme of sGC is in a unique environment. Characterization of the heme environment is critical to the understanding of the mechanism of NO activation. To approach this goal, the beta1 N-terminal fragment consisting of residues 1-385 [beta1(1-385)] of sGC was expressed in E. coli. beta1(1-385) was then purified to homogeneity in two steps by DEAE ion exchange and gel filtration chromatography. Purified beta1(1-385) was found to contain a stoichiometric amount of heme. The UV-visible spectrum of beta1(1-385) is almost identical to that of the native heterodimeric sGC purified from bovine lung. beta1(1-385) binds both NO and CO, leading to a shift in the Soret maximum from 431 nm to 398 and 423 nm, respectively. These spectral shifts are identical to those observed with heterodimeric sGC purified from bovine lung. These results suggest that the heme in the beta1(1-385) is similar to that in the heterodimeric sGC. Therefore, for the first time, the heme binding region of sGC has been unambiguously localized to the N-terminal region of the beta1 subunit. Our data also suggest that the N-terminal region of the beta1 subunit of sGC is itself sufficient for heme binding.     

Structure of the heme d of Penicillium vitale and Escherichia coli catalases

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.     

Effect of various sources of phosphorus on the apparent digestibility of crude nutrients, growing and slaughtering performance as well as selected parameters of metabolism in fattening bulls

Polarized IR measurements on single crystals of human hemoglobin, grown under low salt conditions that stabilize the T quaternary structure, allow spectral features to be associated with individual sites within the molecule. Differences between the a- and c-polarized IR spectra in the sulfhydryl stretching region distinguish the contributions of individual Cys residues to the S-H band and lead to an evaluation of possible H-bonding partners for Cys beta-112. Successful modelling of both crystal and solution S-H spectra with component bands having identical frequencies and bandwidths, supports the use of the X-ray structure as a model for the low affinity T-state in solution. Polarization analysis of a crystal partially saturated with CO reveals comparable occupancy of the alpha- and beta-hemes. In the T-state crystal, the C-O bands are broader and lower in frequency than in the R-state solution, and 20% of the CO-ligated beta-subunits adopt an alternate conformation with a 1967 cm(-1) C-O frequency. The latter observation reflects an energetically significant disruption of the distal heme pocket upon CO binding to beta-hemes in the low affinity T-state.     

Electron transfer in tetrahemic cytochromes c3: spectroelectrochemical evidence for a conformational change triggered by heme IV reduction

Electron transfer in tetrahemic cytochromes c3 from Desulfovibrio vulgaris Hildenborough (D.v.H.) and Desulfovibrio desulfuricans Norway (D.d.N.) strains has been investigated by thin layer spectroelectrochemistry with visible absorption, CD, and resonance Raman (RR) monitoring. The observed splitting of the isosbestic point in the Soret absorption band indicates that the electron transfer from the (FeIII)4 state to the (FeII)4 state proceeds via an intermediate species, which corresponds to 25 and 50% reduction for the D.v.H. cyt.c3 and the D.d.N. cyt.c3, respectively. For the latter, a specific CD signal is observed at half-reduction. RR monitoring of the redox process does not reveal multiple splitting of the high-frequency RR bands, at variance with previously published results on the enzymatic reduction of cyt.c3 from Desulfovibrio vulgaris Miyazaki, a cytochrome highly homologous to D.v.H. cyt.c3 [Verma, A.L., Kimura, A., Nakamura, A., Yagi, T., Inoguchi, H., & Kitagawa, T. (1988) J. Am. Chem. Soc. 110, 6617-6623]. The low-frequency RR spectra of the intermediate species differ significantly from the ones calculated from a linear combination of the all-ferric and all-ferrous states, for the same reduction ratio. Frequency shifts of the bending modes of the cysteine and propionate heme substituents are observed, as well as changes specific to each cytochrome; most notable is the activation of two torsional modes in the case of D.d.N. cyt.c3. Comparison of the results obtained for the two cytochromes leads to the conclusion that reduction of heme IV triggers the observed conformational change. This conclusion is supported by the spectroelectrochemical investigation of the mutant D.v.H. cyt.c3 H25M, in which the sixth ligand of heme III, histidine, is replaced by a methionine.     

Solution 1H-NMR structure of the heme cavity in the low-affinity state for the allosteric monomeric cyano-met hemoglobins from Chironomus thummi thummi. Comparison to the crystal structure

Solution 1H-NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S = 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94]. The crystal structure for the low-pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientation, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338]. All heme pocket residues for the low-pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic-induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.     

Properties of human hemoglobins with increased polarity in the alpha- or beta-heme pocket. Carbonmonoxy derivatives

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the beta-globin Val67(E11) or the alpha-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A0 conformer in the IR spectrum, (iii) increased CO dissociation rates. The spectroscopic data indicate that for the carbonmonoxy derivatives, the Val --> Thr mutation has a larger conformational effect on the beta-subunits than on the alpha-subunits. This is at variance with the deoxy derivatives where the conformational modification was larger in the heme pocket of the alpha-subunit (Cupane, A., Leone, M., Militello, V., Friedman, R. K., Koley, A. P., Vasquez, G. P., Brinigar, W. S., Karavitis, M., and Fronticelli, C. (1997) J. Biol. Chem. 272, 26271-26278). These effects are attributed to a different electrostatic interaction between Ogamma of Thr(E11) and the bound CO molecule. Molecular analysis indicates a more favorable interaction of the bound CO with Thr Ogamma in the beta-subunit heme pocket.