Engineering, expression and biochemical characterization of the hemoglobin domain of a Erwinia chrysanthemi flavohemoprotein.
An artificial hemoglobin-like domain has been constructed by engineering the gene coding for the multi-domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over-expressed in Escherichia coli. The holo-protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140-residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto-oxidation which may explain its tight association with a flavin containing reductase domain. A functional model of this hemoglobin is discussed and compared with the X-ray structures of other hemoproteins.
Labesse
G
G
Département de modélisation des macromolécules biologiques, Laboratoire de Minéralogie-Cristallographie, URA CNRS 09, Universités Paris VI and VII, France. Labesse@lmcp.jussieu.fr
Craescu
C T
CT
Mispelter
J
J
Chottard
G
G
Marden
M C
MC
Pin
S
S
Forest
E
E
Mornon
J P
JP
Boccara
M
M
D002942:Circular Dichroism; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000378:metabolism*; D004926:Escherichia coli; D005420:Flavoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010782:Photolysis; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D005420:Flavoproteins; D006420:Hemeproteins; D006454:Hemoglobins; D011994:Recombinant Proteins