Expression of Pseudomonas stutzeri Zobell cytochrome c-551 and its H47A variant in Escherichia coli.
The nirM gene encoding cytochrome c-551 from Pseudomonas stutzeri Zobell (PZ) has been expressed in Escherichia coli at levels higher than those previously reported but only under strict anaerobic growth conditions. Expression yields for wild-type cytochrome in this study typically reached 0.6 micromol per liter of saturated E. coli culture (5.5mg/L). Culture conditions investigated are compared to obtained c-551 expression levels; the results may lead to a greater understanding of the challenges encountered when expressing c-type hemoproteins in E. coli. The nirM gene was mutated to produce a histidine-47-alanine mutation of c-551 that been heterologously expressed in E. coli using optimum culture conditions and had its physiochemical properties compared to those of the wild-type protein. In PZ, the histidine-47 residue is part of a conserved hydrogen-bonding network located at the bottom of the heme crevice that also involves tryptophan-56 and a heme propionate. Ionization events within this network are experimentally demonstrated to modulate c-551 oxidation-reduction potential and its observed dependence on pH around neutrality. The redox potential of the mutant cytochrome still displays pH-dependence; however, the midpoint potential is approximately 25mV lower with respect to wild-type c-551 at neutral pH while the pK at which the heme propionate (HP-17) ionizes is lowered by 1.3 pH units. Temperature and chemical denaturant studies also show that loss of the hydrogen-bond-donating imidazole leads to a large decrease in c-551 tertiary stability.
Miller
Gregory T
GT
Department of Chemistry, Southern Oregon University, Ashland, OR 97520, USA. millergr@sou.edu
Mackay
Donald Q
DQ
Standley
Melissa S
MS
Fields
Sherry L
SL
Clary
Wendi M
WM
Timkovich
Russell
R
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006639:Histidine / Q000235:genetics; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D006639:Histidine; C017167:cytochrome C(551); D019791:Guanidine; D000409:Alanine