Hydroxide Rather Than Histidine Is Coordinated to the Heme in Five-coordinate Ferric Scapharca inaequivalvisHemoglobin

Abstract

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.The homodimeric hemoglobin (HbI) 1 isolated from arcid clam Scapharca inaequivalvis possesses unique structural features (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Although HbI has a low sequence homology with tetrameric mammalian hemoglobins (Hb), it has a conserved globin fold. It displays a unique structural basis for cooperative ligand binding in that the two hemes face each other across the intersubunit contact and are able to communicate directly through their propionate groups (2,4,15,16). As a consequence of this unusual structural linkage, cooperativity is accompanied by major tertiary changes in the heme environment with only minor rearrangements of the quaternary structure, in contrast to mammalian Hbs (2, 13). The subunit interface in HbI is formed mostly by the helices E and F, which are solventexposed in tetrameric mammalian Hbs.Although a wealth of structural and functional information is available on the ferrous deoxy and ligand-bound forms of HbI (2-14), little is known about the oxidized form (1). The heme structure in the ferric form undergoes a spin transition that is dependent on pH and salt concentration (17,18). From our earlier studies by optical and resonance Raman spectroscopy, it was shown that a mixture of three distinct species are populated in the pH range 6 -9 (17, 18). The formation of a sixcoordinate low-spin heme is favored at acidic pH and high ionic strength and is accompanied by the reversible dissociation of the HbI dimer into monomers. This species is likely to be formed by coordination of the distal histidine to the heme (in addition to the proximal histidine) as shown by EPR studies (18). At neutral pH values, a dimeric pentacoordinate species appears and becomes the dominant form at alkaline pH and low salt. A population of dimeric six-coordinate high-spin heme exists over the entire 6 -9 pH-range and in both low and high salt. Thi...

Giant Extracellular Hemoglobin of Glossoscolex paulistus: An excellent Prototype of Biosensor and Blood Substitute

“…Otzen and Oliveberg (Otzen et al, 2002), studying a small protein S6 in the presence of SDS, argue that monomeric SDS binds to the native state, but global unfolding would occur only above the critical micelle concentration (cmc). Indeed, this verification is corroborated for various works about interaction between surfactants and hemoproteins (Oellerich et al, 2003;Tofani et al, 2004;Das et al, 1999). Oellerich and coworkers (Oellerich et al, 2003), analyzing the interaction between SDS and cytochrome c, explain that the differences observed below and above the cmc are due to the different modes of binding of SDS monomers and micelles.…”

“…In agreement with this discussion, Tofani and co-workers (Tofani et al, 2004) observed for Horse myoglobin (MbH), that only in a SDS/MbH ratio higher than 400, would occur a more significant protein unfolding and, for consequence, a more exposed and accessible heme pocket. Studies focused on the SDS-cytochrome c interaction at neutral pH have demonstrated that the unfolded state is stabilized when occur the bis-histidine (hemichrome) species formation, being that subsequent modifications of the secondary structure are rate-limited by the histidine dissociation rate (Das et al, 1999). Therefore, the hemichrome formation anchors the E helix into the ferric center, and, with the E and F helices maintained connected to the metallic center, the new polypeptide chains arrangement is stabilized, at least partially.…”

The Cytoplasmic Heme-binding Protein (PhuS) from the Heme Uptake System of Pseudomonas aeruginosa Is an Intracellular Heme-trafficking Protein to the δ-Regioselective Heme Oxygenase

“…In conjunction with the visible titration, the acidic 6-c HS form is assigned to the Fe(III)-H 2 O adduct of heme-PhuS, whereas the two 6-c species observed at neutral pH are assigned to 6-c LS and 6-c HS heme hydroxide adducts. The 5-c HS heme form observed at pH 7.2 and above could have either a histidine or a hydroxide as the fifth heme ligand; there is precedent for both (33)(34)(35). The persistence of 6-c LS heme at acidic pH suggests that the acid-base transitions involving an exogenous axial ligand do not account for all of the heme.…”

Estudo espectroscópico do equilíbrio entre espécies hexacoordenada e pentacoordenada dos monômero d nativo e reconstituído da hemoglobina extracelular gigante de Glossoscolex paulistus em meio alcalino

“…Entretanto, a hemoglobina Scapharca inaequivalvis também mostra uma forte dependência do pH, quando da formação de uma espécie pentacoordenada, mesmo contendo a histidina na posição distal 33 . Esta informação é de grande relevância para o presente trabalho, uma vez que o trabalho de Madura e colaboradores 15 estabeleceu que as hemoglobinas de L. terrestris e Scapharca inaequivalvis apresentam significativa similaridade em termos de estrutura secundária.…”

“…De fato, sabendo-se que alguns trabalhos correlacionam a formação de hemicromo à ocorrência de determinados canais de acesso ao solvente 31 , este fenômeno poderia ser, a priori, simplesmente uma mudança conformacional, sem uma perda mais expressiva das estruturas secundária e terciária da proteína. Mesmo em trabalhos envolvendo as duas mais novas globinas descobertas em vertebrados, neuroglobina e citoglobina, a formação de hemicromos e seu estudo espectroscópico tem sido de grande importância, visando a compreensão das propriedades dessas hemoproteínas 32,33 . Neste sentido, vale lembrar que Blumberg e Peisach consideram a transição de spin que leva à formação do hemicromo como uma conseqüência da quebra de ligações hidrofóbicas 34 , o que implicaria, de qualquer maneira, em um maior ou menor rearranjo, enfatizando, uma vez mais, a correlação entre mudanças na primeira esfera de coordenação e alterações conformacionais das cadeias polipeptídicas.…”