Nitric Oxide Binding Properties of Neuroglobin: A CHARACTERIZATION BY EPR AND FLASH PHOTOLYSIS

Sabine Van Doorslaer,Sylvia Dewilde,Laurent Kiger,Sergiu V Nistor,Etienne Goovaerts,Michael C Marden,Luc Moens

doi:10.1074/jbc.m210617200

Abstract

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.

Highlights

Neuroglobin is a recently discovered member of the globin superfamily
Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O2 concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe2؉-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO
NO Binding to Ngb—Fig. 1A shows the Electron paramagnetic resonance (EPR) spectra of E. coli cell cultures overexpressing recombinant wt Ngb and recombinant mutant Ngb (E7-Val, E7-Gln, and E7-Leu)

Summary

EXPERIMENTAL PROCEDURES

Expression Cloning and Purification of Recombinant Wild Type and Mutant Mouse Ngb—Expression cloning and purification of wt and mutant mouse Ngb was performed as described previously [9]. The spectra of the Ngb directly within the E. coli cell culture were measured with the DW2a spectrophotometer (Aminco) in the split beam mode, ranging from 350 to 650 nm. The typical sample concentration was 10 ␮M on a heme basis, and the measurements were performed in a 4 ϫ 10-mm quartz cuvette at 298 K. 1. X-band EPR spectra of samples of E. coli cell cultures overexpressing recombinant wild-type and mutant mouse neuroglobin taken before (A) and after (B) sonication. The sample was equilibrated with 0.009 atm of NO before the reduction of the oxidized fraction with a slight excess of sodium dithionite. Once the heme is bound to NO the samples were kept on ice. The Ngb-NO samples were diluted into the optical cuvette containing the buffered solution in the presence of 1 mM sodium dithionite equilibrated under 1 atm of CO. To correct for baseline drifts, full spectra were measured, and analysis was performed on the spectral differences

RESULTS

C Oxidase

DISCUSSION