Isolation and characterization of the cytochrome domain of flavocytochrome b2 expressed independently in Escherichia coli.
The cytochrome domain of flavocytochrome b2 (L-lactate dehydrogenase) was expressed in the bacterium Escherichia coli and a purification procedure was developed. When expressed in E. coli, the b2-cytochrome domain contains protohaem IX and has an electronic absorption spectrum identical with that of the cytochrome b2 'core' produced by proteolytic cleavage of the enzyme isolated from yeast. The b2-cytochrome domain isolated from E. coli has an Mr of 10,500 and a redox potential of -31 +/- 2 mV. High-field n.m.r. studies indicate pKa values for the haem propionate groups to be 4.8 and 4.6, consistent with these groups being exposed to solvent rather than buried inside the protein. Using n.m.r. spectroscopy, we have determined an electron self-exchange rate constant for the b2-cytochrome domain of 2.3 x 10(6) M-1.s-1, which is more than two orders of magnitude larger than the value obtained for microsomal cytochrome b5, a homologue of b2-cytochrome domain.
Brunt
C E
CE
Department of Chemistry, University of Edinburgh, Scotland, U.K.
Cox
M C
MC
Thurgood
A G
AG
Moore
G R
GR
Reid
G A
GA
Chapman
S K
SK
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification*; D042844:L-Lactate Dehydrogenase (Cytochrome); D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013053:Spectrophotometry
D003062:Codon; D046911:Macromolecular Substances; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)