Neuroglobin and cytoglobin: two new members of globin family

The globin family has long been defined by myoglobin and hemoglobin, proteins with the functions of oxygen storage and transportation, respectively. Recently, two new members of this family were discovered: neuroglobin present in neurons and retinal cells and cytoglobin found in various types of tissue. The increased expression of these proteins in hypoxic conditions first suggested a role in oxygen supply. However structural and functional differences, such as the hexacoordinated heme, a high autoxidation rate and different concentrations between different cellular types, have dismissed this hypothesis. The protective role of these globins has already been established. In vitro and in vivo studies have demonstrated increased survival of neurons under stress in the presence of neuroglobin and increased resistance to neurodegenerative diseases. However the mechanism remains unknown. Functions, including detoxification of nitric oxide, free radical scavenging and as an antioxidant and signaling of apoptosis, have also been suggested for neuroglobin and an antifibrotic function for cytoglobin.

O papel das Argininas alfa-92 e alfa-141 na regulação das propriedades funcionais de hemoglobinas por íons cloreto / The role of alpha-92 and alpha-141 arginines in the hemoglobin functional properties regulated by chloride ions

A oxigenação da Hb humana (HbA) requer um modelo detrês estado: dois estados desoxi To e Tx, livre e complexado comanions, respectivamente e um estado R oxi. A regulação entre essesestados é modulada pela presença de anions, tais como cloreto,que se ligam ao estado T. A ligação de cloreto, entretanto, permanececontroversa. O objetivo deste trabalho foi o estudo dasargininas 92α (interface α1β2) e 141α (C-terminal) como sítios deligação de cloreto. Para isso, estudamos Hbs com mutações sitiodirigidas: mutantes naturais Hb J Cape Town (R92Q), desArg(R141Δ), Chesapeake (R92L) e a construída Chesapeake desArg(R92L,141Δ). As Hbs foram expressas em Escherichia coli epurificadas. Através de curvas de oxigenação, medimos afinidade ecooperatividade, variando a atividade de água e efeito Bohr napresença e ausência de cloreto. Características estruturais foramobtidas por espectroscopia 1H RMN. Parâmetros de oxigenação eEfeito Bohr medidos indicaram uma afinidade maior e cooperatividademenor na ausência e presença de cloreto para todosos mutantes. Modificações estruturais representaram aspectosfuncionais das Hbs modificadas, como aumento significativo naafinidade e mudanças na cooperatividade. Estudos da atividade deágua em função da concentração de cloreto revelaram que a HbdesArg foi a única Hb modificada que seguiu o modelo de trêsestados. As demais não apresentaram o estado Tx, fato confirmadopelo número de moléculas de água ligadas durante a transiçãodesoxi-oxi. Este comportamento sugere que a Arginina 92 poderiaser responsável pela ligação de cloreto, uma vez que a oxigenaçãodas Hbs sem esse resíduo não pode ser ajustada pelo modelo detrês estados. Contudo, o efeito Bohr mostrou que todas as Hbsmodificadas liberaram ~ 1 próton na presença de cloreto, diferenteda HbA que libera ~ 2, sugerindo um papel para arginina 141 noefeito Bohr terciário e quaternário.

The oxygenation of human Hb (HbA) demands a three statemodel: two deoxy states To and Tx, free and complexed withanions respectively, and an oxy R state. The regulation betweenthese states is modulated by the presence of anions, such as chloride,that binds to T state. The binding of chloride, however, remainscontroversial. The aim of this work is the study of arginines 92a(a1ß2 interface) and 141a (C-terminal ) as chloride binding sites.To investigate that, we have studied 92 and 141 site directedmutant species: natural mutants Hb J-Cape-Town (R92Q), desArg(R141Δ), Chesapeake (R92L), and the constructed ChesapeakedesArg (R92L,141Δ). We expressed Hbs in Escherichia coli andpurified. Through oxygen binding curves we measured affinityand cooperativity, in function of water effect and Bohr effect inpresence and absence of chloride. Structural features were obtainedthrough 1H NMR spectroscopy Oxygen binding properties andBohr effect measured indicated a higher affinity and lowercooperativity in absence and presence of chloride for all mutants.Structural changes represent functional aspects of mutant Hbs such as a significant rise in affinity or a change in cooperativity.Water activity studies conducted as a function of chlorideconcentration showed that the only Hb desArg follows the threestate model. The other mutant Hbs do not exhibit the Tx state, afact confirmed by the number of water molecules bound to eachHb during the deoxy-oxy transition. This behavior suggests thatthe Arginine 92 site could be responsible for chloride binding toHb, since oxygenation of 92 mutant Hbs cannot be adjusted bythe three state model. However, Bohr effect showed that all mutantHbs released~1 proton in chloride presence, different from HbAthat releases ~2, suggesting a role for 141 arginine in the tertiaryand quaternary Bohr effect.