The structural bases for the unique ligand binding properties of Glycera dibranchiata hemoglobins. A resonance Raman study

The hemoglobin of the marine annelid Glycera dibranchiata possesses several unique features: the hemoglobin consists of multiple monomeric and polymeric components, quaternary structure is lacking, the distal histidine is replaced by leucine in at least one monomeric constituent, and 4) the protein exhibits extremely rapid ligand binding kinetics. The effect of these structural modifications on the ligand binding process has been evaluated using resonance Raman spectroscopy to examine the vibrational modes of the porphyrin macrocycle in deoxy and carbonmonoxy equilibrium species of hemoglobin G. dibranchiata in both the unseparated monomeric and polymeric forms and in a single monomeric component designated Fraction II. Significant differences relative to hemoglobin were found in porphyrin pi electron density, vinyl environment, low frequency vibrational modes, and, in particular, the Fe-proximal histidine stretching mode. Spectra of the deoxy heme transients generated within 10 ns of ligand photolysis have also been examined. These clearly indicate large differences in the heme pocket dynamics subsequent to CO photolysis in G. dibranchiata hemoglobins relative to other hemoglobins. The significance of these results in terms of the kinetics and thermodynamics of ligand binding is discussed.     

Resonance Raman Spectra of Photodissociated Hemoglobins: Implications on Cooperative Mechanisms

Resonance Raman spectra at cryogenic temperatures of photodissociated hemoglobins and the corresponding deoxygenated preparations are compared and significant differences are found in modes with contributions from peripheral substituents of the heme as well as in the iron-histidine stretching mode. These differences in heme vibrational spectra reflect differences in the tertiary structure of the heme pocket between deoxyhemoglobin and the CO-bound form. An analysis of the effects of cooperative energy storage on the tertiary structure around the heme is made and used to interpret this resonance Raman data. The differences between the spectra of the deoxygenated preparations and the photoproducts provide evidence that a fraction of the free energy of cooperativity, ΔG, is located away from the heme. These data support models for cooperativity in which the cooperative energy is distributed over many bonds or is localized in protein bonds only, such as those at the subunit interface. In addition, the local changes in amino acid positions, which must occur following the change in the state of ligand binding, may drive the changes in the structural relationships of the subunits and hence form one of the initial steps for triggering the quaternary structure transition.     

Different roles of protein dynamics and ligand migration in non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana

The ligand rebinding kinetics after photolysis of the CO complexes of Arabidopsis thaliana hemoglobins AHb1 and AHb2 in solution show very different amplitudes in the geminate phase, reflecting different migration pathways of the photodissociated ligand in the system of internal cavities accessible from the heme. The dependence of the geminate phase on CO concentration, temperature, encapsulation in silica gels and presence of glycerol confirms a remarkable difference in the internal structure of the two proteins and a dramatically different role of protein dynamics in regulating the reactivity with CO. This finding strongly supports the idea that they have distinct physiological functions.     

Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy-entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.     

Kinetics of ligand binding and quaternary conformational change in the homodimeric hemoglobin from Scapharca inaequivalvis

The kinetics of the reaction with oxygen and carbon monoxide of the homodimeric hemoglobin from the bivalve mollusc Scapharca inaequivalvis has been extensively investigated by flash and dye-laser photolysis, temperature jump relaxation, and stopped flow methods. The results indicate that cooperativity in ligand binding, already observed for oxygen at equilibrium, finds its kinetic counterpart in a large decrease of the oxygen dissociation velocity in the second step of the binding reaction. In the case of carbon monoxide, cooperativity is clearly evident in the increase of the combination velocity constant as the reaction proceeds. Therefore, the ligand-binding kinetics of this dimeric hemoglobin shows the characteristic features of the corresponding reactions of tetrameric hemoglobins. Analysis of the data in terms of the allosteric model proposed by Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118) has shown that the values of the allosteric parameters cannot be fixed uniquely for a dimeric hemoglobin. The rapid changes in absorbance observed at the isosbestic points of unliganded and liganded hemoglobin following laser photolysis provided a value of 7 X 10(4) S-1 at 20 degrees C for the rate of the ligand-free quarternary conformational change, postulated on the basis of cooperative ligand binding. Comparison of the rapid absorbance changes observed during ligand rebinding in this hemoglobin with those observed in tuna hemoglobin indicate that, at full photolysis, binding to the T state is followed by further binding and conversion to the liganded R state; at partial photolysis, population of the liganded T state occurs immediately and is followed by a decay to the liganded R state upon further ligand binding. These new results, in conjunction with previous equilibrium data on the same system, show unequivocally that the presence of two different types of chain is not an absolute prerequisite for cooperativity in hemoglobins, contrary to currently accepted ideas.     

Complex interactions of carbon monoxide with reduced cytochrome cbb3 oxidase from Pseudomonas stutzeri

Cytochrome cbb(3) oxidase, from Pseudomonas stutzeri, contains a total of five hemes, two of which, a b-type heme in the active site and a hexacoordinate c-type heme, can bind CO in the reduced state. By comparing the cbb(3) oxidase complex and the isolated CcoP subunit, which contains the ligand binding bishistidine-coordinated c-type heme, we have deconvoluted the contribution made by each center to CO binding. A combination of rapid mixing and flash photolysis experiments, coupled with computer simulations, reveals the kinetics of the reaction of c-type heme with CO to be complex as a result of the need to displace an endogenous axial ligand, a property shared with nonsymbiotic plant hemoglobins and some heme-based gas sensing domains. The recombination of CO with heme b(3), unlike all other heme-copper oxidases, including mitochondrial cytochrome c oxidase, is independent of ligand concentration. This observation suggests a very differently organized dinuclear center in which CO exchange between Cu(B) and heme b(3) is significantly enhanced, perhaps reflecting an important determinant of substrate affinity.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species

Hexacoordinate hemoglobins are found in many living organisms ranging from prokaryotes to plants and animals. They are named "hexacoordinate" because of reversible coordination of the heme iron by a histidine side chain located in the heme pocket. This endogenous coordination competes with exogenous ligand binding and causes multiphasic relaxation time courses following rapid mixing or flash photolysis experiments. Previous rapid mixing studies have assumed a steady-state relationship between hexacoordination and exogenous ligand binding that does not correlate with observed time courses for binding. Here, we demonstrate that this assumption is not valid for some hexacoordinate hemoglobins, and that multiphasic time courses are due to an appreciable fraction of pentacoordinate heme resulting from relatively small equilibrium constants for hexacoordination (K(H)). CO binding reactions initiated by rapid mixing are measured for four plant hexacoordinate hemoglobins, human neuroglobin and cytoglobin, and Synechocystis hemoglobin. The plant proteins, while showing a surprising degree of variability, differ from the others in having much lower values of K(H). Neuroglobin and cytoglobin display dramatic biphasic time courses for CO binding that have not been observed using other techniques. Finally, an independent spectroscopic quantification of K(H) is presented that complements rapid mixing for the investigation of hexacoordination. These results demonstrate that hexacoordination could play a much larger role in regulating affinity constants for ligand binding in human neuroglobin and cytoglobin than in the plant hexacoordinate hemoglobins.     

A model for ligand binding to hexacoordinate hemoglobins

Hexacoordinate hemoglobins are heme proteins capable of reversible intramolecular coordination of the ligand binding site by an amino acid side chain from within the heme pocket. Examples of these proteins are found in many living organisms ranging from prokaryotes to humans. The nonsymbiotic hemoglobins (nsHbs) are a class of hexacoordinate heme proteins present in all plants. The nsHb from rice (rHb1) has been used as a model system to develop methods for determining rate constants characterizing binding and dissociation of the His residue responsible for hexacoordination. Measurement of these reactions exploits laser flash photolysis to initiate the reaction from the unligated, pentacoordinate form of the heme protein. A model for ligand binding is presented that incorporates the reaction following rapid mixing with the reaction starting from the pentacoordinate hemoglobin (Hb). This model is based on results indicating that ligand binding to hexacoordinate Hbs is not a simple combination of competing first order (hexacoordination) and second order (exogenous ligand binding) reactions. Ligand binding following rapid mixing is a multiphasic reaction displaying time courses ranging from milliseconds to minutes. The new model incorporates a "closed", slow reacting form of the protein that is not at rapid equilibrium with the reactive conformation. It is also demonstrated that formation of the closed protein species is not dependent on hexacoordination.     

Transient Raman study of horseradish peroxidase. Evidence for a lack of extensive heme pocket relaxation subsequent to carbon monoxide photolysis

Time-resolved resonance Raman spectroscopy is a valuable tool for the study of the dynamics of heme-protein interactions. In particular, the photolysis of a ligand by short laser pulses allows for the examination of the dynamic evolution of heme-protein interactions subsequent to ligand dissociation. To date, such studies have been confined largely to hemoglobins and myoglobins. Here we present the results of the first transient Raman study of a peroxidase. Resonance Raman spectra of horseradish peroxidase were obtained within 10 ns of ligand (CO) photolysis at a variety of pH values. We find that there is only minimal relaxation of the heme pocket of horseradish peroxidase in response to ligand photolysis. This relaxation is pH-dependent and most probably involves the heme vinyl substituents. Such behavior is in sharp contrast to the transient behavior of most hemoglobins and beef heart cytochrome oxidase.     

A cooperative oxygen binding hemoglobin from Mycobacterium tuberculosis. Stabilization of heme ligands by a distal tyrosine residue

The homodimeric hemoglobin (HbN) from Mycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O(2), CO, and OH(-) derivatives of the wild type protein and the B10 Tyr --> Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.     

Variable Subunit Contact and Cooperativity of Hemoglobins

Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact. Received: 12 January 2001 / Accepted: 3 April 2001     

Spectroscopic and kinetic properties of an oxygen-binding heme protein from Chromatium vinosum

Resonance Raman and electron paramagnetic resonance spectroscopy have been utilized to identify histidine as an axial heme ligand in a high spin, heme c-containing protein isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum. Resonance Raman spectroscopy has also been used to characterize the CO adduct of the C. vinosum hemoprotein. Resonance Raman spectra of the heme site obtained within 10 ns of CO photolysis from the ferrous hemoprotein are virtually identical to those of the unligated protein, indicating that there is little or no rearrangement of the heme pocket in response to ligand photolysis. The equilibrium constant for CO binding to the ferrous hemeprotein was measured to be 1.7 X 10(-5) M-1 and the CO association rate constant determined to be 5.4 X 10(3) M-1 S-1. The quantum efficiency for photodissociation of the hemoprotein X CO complex was greater than or equal to 0.9.     

NMR studies of the heme pocket conformations of monomeric hemoglobins from Glycera dibranchiata. Implications for ligand binding

Two-dimensional 1H-NMR methods have been used to assign side-chain resonances for the tryptophan residues and for several amino acids located in the heme pockets of the carbon monoxide complexes of the major monomeric hemoglobins from Glycera dibranchiata. The NMR spectra reveal a high degree of conservation of the heme pocket structure in the different hemoglobins. However some conformational differences are evident and residues at positions B10 and G8 on the distal side of the heme pocket are not conserved. From the present NMR studies it appears that the monomeric G. dibranchiata hemoglobin examined by X-ray crystallography [Padlan, E. A. & Love, W. (1974) J. Biol. Chem. 249, 4067-4078] corresponds to HbC. Except that the orientation of the heme in solution is the reverse of that reported in the crystal structure, there is a close correspondence between the heme pocket structure in the crystal and in solution. The proximal histidine coordination geometry is almost identical in the CO complexes of the three monomeric hemoglobins studied. Distal residues are strongly implicated in determining the observed kinetic differences in ligand binding reactions. In particular, steric crowding of the ligand binding site in hemoglobin A is probably a major factor in the slower kinetics of this component.     

Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy and infrared spectroscopy have been used to characterize the three vibrational modes, CO and FeC stretching and FeCO bending, for carbon monoxide bound to reduced horseradish peroxidase, with the aid of 13CO and C18O isotope shifts. At high pH, one species, I, is observed, with nu FeC = 490 cm-1 and nu CO = 1932 cm-1. The absence of a band attributable to delta FeCO suggests a linear FeCO unit normal to the heme plane. The data were consistent with I having a strongly H-bonded proximal histidine, as shown by a comparison with imidazole and imidazolate adducts of FeIIPPDME(CO) (PPDME = protoporphyrin IX dimethyl ester), with nu FeC = 497 and 492 cm-1 and nu CO = 1960 and 1942 cm-1. At low pH an additional species, II, is observed, with nu FeC = 537 cm-1, nu CO = 1904 cm-1, and delta FeCO = 587 cm-1; it is attributed to FeCO that is H bonded to a protonated distal histidine, the H bond strongly lowering nu CO and raising nu FeC. The appearance of delta FeCO in the RR spectrum suggests that the FeCO unit in II is tilted with respect to the heme plane. At low pH, the population of I and II depends on the CO concentration. I dominates at low CO/protein levels but is replaced by II as the amount of CO is increased. This behavior is suggested to arise from secondary binding of CO, which induces a conformation change involving the distal residues of the heme pocket.     

Absence of cooperative energy at the heme in liganded hemoglobins

Using resonance Raman and infrared absorption spectroscopies, we show that there are no energetically significant structural changes at the heme upon the quaternary structure transition in six-coordinate hemoglobins. These observations are at variance with the presently accepted mechanism for cooperativity, which postulates severe strain in the T quaternary structure of liganded hemoglobin. By consideration of the present results, and studies on deoxyhemoglobins and photodissociated hemoglobins, a view of the distribution of the free energy of cooperativity emerges. In five-coordinate deoxyhemoglobins the iron-histidine bond is able to respond to the protein structure, thereby accounting for a wide variation (40 cm(1] in its frequency. In contrast, when a sixth ligand is present and the iron is pulled into plane, the histidine-heme-ligand complex becomes structurally rigid, thereby preventing protein-induced changes at the heme. Instead, in liganded hemoglobin the changes in structure that occur at the subunit interface upon the quaternary structure transition are accommodated away from the heme by relatively weak bonds in the protein.     

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.     

Mycobacterium tuberculosis KatG(S315T) catalase-peroxidase retains all active site properties for proper catalytic function

Mycobacterium tuberculosis (Mtb) KatG is a catalase-peroxidase that is thought to activate the antituberculosis drug isoniazid (INH). The local environment of Mtb KatG and its most prevalent INH-resistant mutant, KatG(S315T), is investigated with the exogenous ligands CO and NO in the absence and presence of INH by using resonance Raman, FTIR, and transient absorption spectroscopy. The Fe-His stretching vibration is detected at 244 cm(-)(1) in the ferrous forms of both the wild-type enzyme and KatG(S315T). The ferrous-CO complex of both enzymes exhibits nu(CO), nu(Fe-CO), and delta(Fe-C-O) vibrations at 1925, 525, and 586 cm(-)(1), respectively, indicating a positive electrostatic environment for the CO complex, which is probably weakly hydrogen-bonded to a distal residue. The CO geometry is nonlinear as indicated by the unusually high intensity of the Fe-C-O bending vibration. The nu(Fe(III)-NO) and delta(Fe(III)-N-O) vibrations are detected at 596 and 571 cm(-)(1), respectively, in the ferric forms of wild-type and mutant enzyme and are indicative of a nonlinear binding geometry in support of the CO data. Although the presence of INH does not affect the vibrational frequencies of the CO- and NO-bound forms of either enzyme, it seems to perturb slightly their Raman intensities. Our results suggest a minimal, if any, perturbation of the distal heme pocket in the S315T mutant. Instead, the S315T mutation seems to induce small changes in the KatG conformation/dynamics of the ligand access channel as indicated by CO rebinding kinetics in flash photolysis experiments. The implications of these findings for the catalytic mechanism and mechanism of INH resistance in KatG(S315T) are discussed.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.