The Unique Heme–Heme Interactions of the HomodimericScapharca inaequivalvisHemoglobin as Probed in the Protein Reconstituted with Unnatural 2,4 Heme Derivatives (original) (raw)
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Journal of Biological Chemistry, 1999
The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6 -9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm ؊1 and shifts to 553 cm ؊1 in H 2 18 O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.
Unusual Rocking Freedom of the Heme in the Hydrogen Sulfide-Binding Hemoglobin from Lucina pectinata
Journal of the American Chemical Society, 1998
Hemoglobin I (HbI) from the clam Lucina pectinata is, in its natural environment, a hydrogen sulfide (H 2 S)-transport heme protein. The resonance Raman (RR) spectrum of the metaquo and deoxyHbI species shows a very weak intensity peak at 370 cm-1 that corresponds to the normal mode of the heme propionates. This suggests the presence of a moderate hydrogen bonding between Arg99 and the heme-7propionate. However, the RR spectra of the metcyano, carbonmonoxy, and oxy HbI derivatives reveal the absence of the propionate vibrational frequency at 370 cm-1. The mode is insensitive to the oxidation state of the heme iron, but disappears when the HbI-ligand moiety is formed. These results propose the existence of flexible propionate groups which can result in a weaker hydrogen bond upon heme ligand binding. The longitudinal relaxation time (T 1) 1 H NMR data for the paramagnetic metcyano complex of HbI suggested that the 17.90 ppm signal belongs to the heme-6-propionate R′ protons (6-H R′). In relation to other myoglobins, the large difference in chemical shifts of this signal is attributed both to the lack of hydrogen bonds between the heme-6-propionate group and amino acid residues and to a flexible orientation of the side chain with respect to the heme plane. The data predict a model where the heme group of HbI is tightly bound to His96 (ν Fe-His at 218 cm-1), but due to the absence of strong hydrogen bonding interactions between the heme propionates and the nearby amino acids, the heme is not firmly anchored. Thus, relative to other heme proteins, the heme group of HbI from Lucina pectinata presents a rocking freedom that facilitates the binding between the heme and the incoming ligand.
Ligand binding to heme proteins: II. Transitions in the heme pocket of myoglobin
Biophysical Journal, 1993
Phenomena occurring in the heme pocket after photolysis of carbonmonoxymyoglobin (MbCO) below about 100 K are investigated using temperature-derivative spectroscopy of the infrared absorption bands of CO. MbCO exists in three conformations (A substates) that are distinguished by the stretch bands of the bound CO. We establish connections among the A substates and the substates of the photoproduct (B substates) using Fourier-transform infrared spectroscopy together with kinetic experiments on MbCO solution samples at different pH and on orthorhombic crystals. There is no one-to-one mapping between the A and B substates; in some cases, more than one B substate corresponds to a particular A substate. Rebinding is not simply a reversal of dissociation; transitions between B substates occur before rebinding. We measure the nonequilibrium populations of the B substates after photolysis below 25 K and determine the kinetics of B substate transitions leading to equilibrium. Transitions between B substates occur even at 4 K, whereas those between A substates have only been observed above about 160 K. The transitions between the B substates are nonexponential in time, providing evidence for a distribution of substates. The temperature dependence of the B substate transitions implies that they occur mainly by quantum-mechanical tunneling below 10 K. Taken together, the observations suggest that the transitions between the B substates within the same A substate reflect motions of the CO in the heme pocket and not conformational changes. Geminate rebinding of CO to Mb, monitored in the Soret band, depends on pH. Observation of geminate rebinding to the A substates in the infrared indicates that the pH dependence results from a population shift among the substates and not from a change of the rebinding to an individual A substate. INTRODUCTION1 Myoglobin is a globular protein consisting of 153 amino acids and a heme (Fe-protoporphyrin IX) as the prosthetic group. Textbooks state that the function of Mb is the reversible binding of small ligands such as dioxygen (02) and carbon monoxide (CO) at the heme iron (Stryer, 1988). One could expect such a binding process to be simple. It was indeed originally described as a one-step process (Antonini and Brunori, 1971). Flash photolysis experiments performed over wide ranges in time and temperature imply, however, that the binding process is far from simple (Austin et al., 1975). Four phenomena, in particular, produce complexity: 1. multiple wells along the reaction coordinate, 2. confor-mational substates, 3. protein relaxation after photodissociation, and 4. thermal fluctuations. In the following, we briefly describe how these phenomena affect the ligand binding to myoglobin. Multiple wells along the reaction coordinate In the simplest model, CO in the solvent binds to the heme iron in one step. Flash photolysis data, however, show evidence for multiple processes. A model that describes the salient features of the kinetic data uses three wells (states) along the reaction coordinate, A i± B S.
Biochimica Et Biophysica Acta-proteins and Proteomics, 2005
The distal pocket of hemoglobin II (HbII) from Lucina pectinata is characterized by the presence of a GlnE7 and a TyrB10. To elucidate the functional properties of HbII, biophysical studies were conducted on HbII and a HbI PheB10Tyr site-directed mutant. The pH titration data at neutral conditions showed visible bands at 486, 541, 577 and 605 nm for both proteins. This suggests the possible existence of a conformational equilibrium between an open and closed configuration due to the interactions of the TyrB10, ligand, and heme iron. The kinetic behavior for the reaction of both ferric proteins with H 2 O 2 indicates that the rate for the formation of the ferryl intermediates species varies with pH, suggesting that the reaction is strongly dependent on the conformational states. At basic pH values, the barrier for the reaction increases as the tyrosine adopts a closed conformation and the ferric hydroxyl replaces the met-aquo species. The existence of these conformers is further supported by resonance Raman (RR) data, which indicate that in a neutral environment, the ferric HbII species is present as a possible mixture of coordination and spin states, with values at 1558 and 1580 cm -1 for the r 2 marker, and 1479, 1492, and 1503 cm À1 for the r 3 mode. Moreover, the presence of the A 3 and A o conformers at 1924 and 1964 cm À1 in the HbII-CO infrared spectra confirms the existence of an open and closed conformation due to the orientation of the TyrB10 with respect to the heme active center. D
The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme
Proteins: Structure, Function, and Genetics, 2002
Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.
Heme-heme interactions in tetramers and dimers of hemoglobin subunits: DeVoe theory calculations
Chirality, 2005
Detectable exciton couplets arising from heme-heme interactions in the hemoglobin (Hb) tetramers of HbO 2 and deoxyHb were predicted by DeVoe theory (Woody, in: Optical properties and structure of tetrapyrroles. Berlin: Walter deGruyter & Co.; 1985. p 239-259). This prediction was supported by the observation of an exciton couplet in the CD difference spectrum between the Hb tetramer and the ab dimer of HbCO (Goldbeck et al., Biochem Biophys Res Commun 1997;235:610-614). In this paper, DeVoe theory is used to calculate the heme-heme interactions in the CO complex of the Hb tetramer (a 2 b 2) and dimer (ab), the systems studied by Goldbeck et al. The couplet strength of the resulting theoretical CD difference spectrum agrees well with experiment, thus confirming that heme-heme interactions contribute significantly to the CD of HbCO. Given that the heme-heme distances in HbCO are 25 Å and more, it is highly likely that heme-heme interactions also contribute significantly to the CD of other multiheme proteins, e.g., cytochrome c 3 , cytochrome oxidase, cytochrome bc 1 , etc., where the hemes are in closer proximity.