Search

Immobilization of redox proteins on electrode surfaces is of special interest for mechanistic studies and applications, because of the well-controlled redox state of protein molecules at a polarized electrode and fast electron-transfer kinetics that are free from diffusion limitation. Here, bis-organosilane [1,2-bis(trimethoxysilyl)ethane] was used as a fresh solution in a pH 7 phosphate buffer without the use of any organic solvent, sol-gel, or mesoporous bulk matrix. A short aging period of 30 min before deposition on the electrodes was optimal for the immobilization of proteins. Three redox proteins (cytochrome c, neuroglobin, and GLB-12) were confined to the gold surface of electrodes with high coverages and stabilities, indicating that the suggested technique is simple, efficient, and generic in nature. [ABSTRACT FROM AUTHOR]

• A nanoporous gold surface enabled rapid direct electron transfer of neuroglobin. • An electrocatalytic signal upon addition of potential substrate was observed. • Electron transfer from ferrous neuroglobin to ferric cytochrome c was detected. • Mutational studies provided insights into anti-apoptotic function of Ngb in cells. Human neuroglobin (Ngb) is a heme protein expressed in the brain tissue and retina with an important neuroprotective role. However, its molecular mechanism is still under exploration. To gain further insight into this issue, we developed an electrochemical strategy using a facilely prepared nanoporous gold (NPG) electrode. Immobilization of Ngb onto the NPG surface enabled rapid direct electron transfer between Ngb and the electrode, whereas less electrochemical response was generated using a planar gold electrode. Voltammograms recorded using the NPG electrodes gave an E 0′ value of −0.170 V (vs. standard hydrogen electrode (SHE)), which is in good agreement with the previously reported values. Furthermore, a heterogeneous electron transfer rate constant of 46 s−1 was obtained, which was larger than the rate constants reported previously. The addition of ferric cytochrome c (Cyt c), a potential redox partner protein of Ngb, increased the cathodic current in the voltammogram, indicating the electron transfer from the reduced (ferrous) Ngb to the ferric Cyt c. In addition, mutational studies demonstrated for the first time that the disulfide bond formation between Cys46 and Cys55 is important for this electron transfer. These results may indicate that ferrous Ngb, in addition to the ferric form, also contributes to the anti-apoptotic properties of Ngb, depending on the disulfide bond formation. Thus, the NPG-based electrochemical system developed in our study is an effective tool for uncovering the molecular basis of Ngb coupled with electron transfer. [ABSTRACT FROM AUTHOR]

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. Mammalian Ngb is involved in neuroprotection under conditions of oxidative stress, such as ischemia and reperfusion. We previously found that zebrafish Ngb can penetrate the mammalian cell membrane. In the present study, we investigated the functional characteristics of fish Ngb by using the zebrafish cell line ZF4 and zebrafish retina. We found that zebrafish Ngb translocates into ZF4 cells, but cannot protect ZF4 cells against cell death induced by hydrogen peroxide. Furthermore, we demonstrated that a chimeric ZHHH Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish, is a cell-membrane-penetrating protein that can protect ZF4 cells against hydrogen peroxide exposure. Moreover, we investigated the localization of Ngb mRNA and protein in zebrafish retina and found that Ngb mRNA is expressed in amacrine cells in the inner nuclear layer and is significantly increased in amacrine cells 3days after optic nerve injury. Immunohistochemical studies clarified that Ngb protein levels were increased in both amacrine cells and presynaptic regions in the inner plexiform layer after nerve injury. Taken together, we hypothesize that fish Ngb, whose expression is upregulated in amacrine cells after optic nerve injury, might be released from amacrine cells, translocate into neighboring ganglion cells, and function in the early stage of optic nerve regeneration. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O2 and facilitates the O2 flux within a myocyte, displays the allosteric modulation of the O2 affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O2 diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)