Abstract
The true function of neuroglobin (Ngb) and, particularly, human Ngb (NGB) has been under debate since its discovery 15 years ago. It has been expected to play a role in oxygen binding/supply, but a variety of other functions have been put forward, including NO dioxygenase activity. However, in vitro studies that could unravel these potential roles have been hampered by the lack of an Ngb-specific reductase. In this work, we used electrochemical measurements to investigate the role of an intermittent internal disulfide bridge in determining NO oxidation kinetics at physiological NO concentrations. The use of a polarized electrode to efficiently interconvert the ferric (Fe(3+)) and ferrous (Fe(2+)) forms of an immobilized NGB showed that the disulfide bridge both defines the kinetics of NO dioxygenase activity and regulates appearance of the free ferrous deoxy-NGB, which is the redox active form of the protein in contrast to oxy-NGB. Our studies further identified a role for the distal histidine, interacting with the hexacoordinated iron atom of the heme, in oxidation kinetics. These findings may be relevant in vivo, for example, in blocking apoptosis by reduction of ferric cytochrome c, and gentle tuning of NO concentration in the tissues.
Highlights
Introduction ofNO into the cell restored the peak current proportional to the NO concentration (Fig. 4, A and B)
The increase in the peak current implies the increase of the steady-state concentration of the ferrous NGB*, which was accumulated because of slow distal histidine dissociation during the oxygen-binding reaction becoming the rate-limiting step of the ongoing reaction (Fig. 5A)
It is noteworthy that NO concentrations higher than 1 M resulted in a partial decrease in the ferrous NGB* fraction, which can be explained by inhibition of the heme by strongly bound NO (Fig. 1, reaction b)
Summary
Electrochemical Behavior of NGB—Under N2 atmosphere, NGB* showed highly reversible electrochemical behavior when either dissolved in solution or immobilized on the electrodes (Fig. 2). Effect of O2 Concentration—The process of O2 binding in NGB is preceded by a relatively slow distal histidine dissociation (k ϭ 7 sϪ1 for NGB* and 0.6 sϪ1 for NGBSS as measured by flash photolysis [34]) as represented in Scheme 1. The increase in the peak current implies the increase of the steady-state concentration of the ferrous NGB*, which was accumulated because of slow distal histidine dissociation during the oxygen-binding reaction becoming the rate-limiting step of the ongoing reaction (Fig. 5A). It is noteworthy that NO concentrations higher than 1 M resulted in a partial decrease in the ferrous NGB* fraction, which can be explained by inhibition of the heme by strongly bound NO (Fig. 1, reaction b).
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