A novel Cu,Zn superoxide dismutase from the fungal strain Humicola lutea 110: isolation and physico-chemical characterization.
The fungal strain Humicola lutea 110 produces a mangan- and a copper zinc-containing superoxide dismutases (SOD). In this study, the purification, N-terminal sequence and spectroscopic properties of the new Cu,Zn SOD are described. The preparation of the pure metalloenzyme was achieved via treatment of the strain with acetone followed by gel and ion exchange chromatography. The protein consists of 302 amino acid residues and has a molecular mass of approximately 32 kDa, as determined by PAG electrophoresis and 3100 U mg-1 protein-specific activity. It is a dimeric enzyme with two identical subunits of 15,950 Da, as indicated by SDS-PAGE, mass spectroscopic and amino acid analysis. The N-terminal sequence analysis of the Cu,Zn SOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Conformational stability and reversibility of unfolding of the dimeric enzyme were determined by fluorescence and circular dichroism (CD) spectroscopy. The critical temperature of deviation from linearity (Tc) of the Arrhenius plot ln (Q-1(-1)) vs. 1/T was calculated to be 68 degrees C and the respective activation energy for the thermal deactivation of the excited indole chromophores is 42 kcal mol-1. The melting temperatures (Tm) were determined by CD measurements to be 69 degrees C for the holo- and 61 degrees C for the apo-enzyme. The fluorescence emission of the Cu,Zn SOD is dominated by 'buried' tryptophyl chromophores. Removal of the copper-dioxygen system from the active site caused a 4-fold increase of the fluorescence quantum yield and a 10 nm shift of the emission maximum position towards higher wavelength.
Dolashka-Angelova
P
P
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria. dolashka@orgchm.acad.bg
Angelova
M
M
Genova
L
L
Stoeva
S
S
Voelter
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006801:Humans; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology
D013482:Superoxide Dismutase