Heme-Containing Oxygenases

TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue

Although phenomenlogical models that account for cooperativity in allosteric systems date back to the early and mid-60's (e.g., the KNF and MWC models), there is resurgent interest in the topic due to the recent experimental and computational studies that attempted to reveal, at an atomistic level, how allostery actually works. In this review, using systems for which atomistic simulations have been carried out in our groups as examples, we describe the current understanding of allostery, how the mechanisms go beyond the classical MWC/Pauling-KNF descriptions, and point out that the “new view” of allostery, emphasizing “population shifts,” is, in fact, an “old view.” The presentation offers not only an up-to-date description of allostery from a theoretical/computational perspective, but also helps to resolve several outstanding issues concerning allostery.

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Allostery and cooperativity revisited.

The majority of modern organisms, including many prokaryotes, are aerobes;1 that is, they use molecular oxygen as the terminal electron acceptor for energy generation. Although nearly every redox gradient in nature appears to be utilized by one organism or another,2-4 aerobic metabolism predominates, in large part due to the highly exergonic nature of the four-electron, four-proton (4e/4H+) reduction of O2 to H2O (reaction 1). A multicellular aerobe requires an

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Functional analogues of cytochrome c oxidase, myoglobin, and hemoglobin.

As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and trans...

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

Human myeloperoxidase and human thyroid peroxidase nucleotide and amino acid sequences were compared. The global similarities of the nucleotide and amino acid sequences are 46% and 44%, respectively. These similarities are most evident within the coding sequence, especially that encoding the myeloperoxidase functional subunits. These results clearly indicate that myeloperoxidase and thyroid peroxidase are members of the same gene family and diverged from a common ancestral gene. The residues at 416 in myeloperoxidase and 407 in thyroid peroxidase were estimated as possible candidates for the proximal histidine residues that link to the iron centers of the enzymes. The primary structures around these histidine residues were compared with those of other known peroxidases. The similarity in this region between the two animal peroxidases (amino acid 396-418 in thyroid peroxidase and 405-427 in myeloperoxidase) is 74%; however, those between the animal peroxidases and other yeast and plant peroxidases are not significantly high, although several conserved features have been observed. The possible location of the distal histidine residues in myeloperoxidase and thyroid peroxidase amino acid sequences are also discussed.

Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family

Confirmation of the assignment of the iron-histidine stretching mode in myoglobin

The origin of the differences in oxygen binding energy in various haemoglobins and myoglobins has long been debated. Perutz1 proposed that the haem-coordinated histidine (proximal histidine) strains the haem iron in low affinity globins but relaxes it in high affinity globins. The existence of such tension in T-structure deoxyhaemoglobin (deoxyHb) was recently confirmed by electron paramagnetic resonance (EPR)2,3, resonance Raman4,5 and NMR6 spectroscopy. Although its contribution to the free energy of cooperativity is insignificant in the deoxy state, the tension at the haem is considered to be ∼1 kcal mol−1 for the ligated form in which the haem iron moves into the porphyrin plane7. The remaining free energy is probably stored in other parts of the molecule. Therefore, a study of the stabilization mechanisms of the oxygenated form became increasingly important. A hydrogen bond between the bound oxygen and the distal histidine has been proposed by Pauling8; this would be expected to stabilize the oxy form of the protein and could contribute to the regulation of the oxygen affinity through the oxygen dissociation rate. A series of EPR and functional studies on various cobalt-substituted monomeric haemoglobins and myoglobins suggested the presence of such hydrogen bonding8–12 and it has recently been established in crystals of oxy iron myoglobin (oxyFeMb)13 and in oxyhaemoglobin14. Here we present resonance Raman spectra of the oxy forms of cobalt–porphyrin-substituted myoglobin and haemoglobin (CoMb and CoHb) recorded in buffered H2O and D2O solutions at 406.7 nm excitation. Only the Raman lines corresponding to the O—O stretching mode of the bound oxygen15, appearing near 1,130 cm−1, are shifted (2–5 cm−1) on replacement of H2O by D2O; no other vibrations, including the Co—O2 stretching mode, exhibit any frequency shifts. This indicates that the bound oxygen in oxyCoMb and in both subunits of oxyCoHb interacts with the adjacent exchangeable proton, and confirms the formation of a hydrogen bond between the bound oxygen and the distal histidine9.

Evidence for hydrogen bonding of bound dioxygen to the distal histidine of oxycobalt myoglobin and haemoglobin

"Time-resolved Raman studies have shown that communication between the heme oxygen binding sites and the surrounding globin occurs through the iron-proximal histidine linkage. By comparing the frequency of the Fe-His stretching mode in equilibrium deoxy- and photoinduced transient deoxyhemoglobins, we have found that ligand binding induces protein structural changes that strengthen the Fe-His linkage. The extent of this increase is observed to depend upon the quaternary state. This dependence is reflected in the restricted set of values observed in transient and equilibrium studies for the frequency of the Fe-His stretching mode in both R and T state deoxyhemoglobins. For T state hemoglobins the frequency increases from 215 cm" to -222 cm" in going from the stable to the nanosecond transient deoxy species. The corresponding change for R state human hemoglobins is from -222 cm" to "230 cm". Studies on transients derived from Fe-Co hybrid hemoglobins reveal no subunit heterogeneity associated with the R state transient (230 cm") at high pH. We also observe that perturbations of the protein known to destabilize the structure of ligand-bound R state hemoglobins are associated with a decrease in the frequency of the iron-proximal histidine stretching mode in the corresponding transient species. Such effects can originate from changes in either quaternary state or solution conditions as well as species-specific variations in protein structure. These results indicate that modulation of the Fe-His linkage could be a general mechanism for regulating ligand binding properties in hemoglobin. A direct connection between ligand binding and the Fe-His bond is suggested from our finding that the structural parameter(s) regulating the barrier height for geminate recombination is likely to be the same as the one(s) modulating the frequency of the Fe-His stretching mode in the transient deoxy species. Based on the recent conclusion that the variation in the tilt of histidine with respect to the heme plane is primarily responsible for the spectrum of frequencies observed for the Fe-His stretching mode we have examined a model in which

Masao Ikeda-Saito

Papers

Human myeloperoxidase and thyroid peroxidase, two enzymes with separate and distinct physiological functions, are evolutionarily related members of the same gene family

Confirmation of the assignment of the iron-histidine stretching mode in myoglobin

Evidence for hydrogen bonding of bound dioxygen to the distal histidine of oxycobalt myoglobin and haemoglobin

The Iron-proximal Histidine Linkage and Protein Control of Oxygen Binding in Hemoglobin

Thermodynamic properties of oxygen equilibria of dimeric and tetrameric hemoglobins from Scapharca inaequivalvis