Laser photolysis behavior of ferrous horseradish peroxidase with carbon monoxide and cyanide: effects of mutations in the distal heme pocket.
Native horseradish peroxidase and several forms with mutations in the distal heme pocket (His42Leu, His42Arg, and Arg38Leu) have been expressed in Escherichia coli. These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxide and cyanide after photolysis of the reduced forms. The recombinant wild-type ferrous form exhibited monophasic recombination of carbon monoxide with an observed bimolecular rate constant at pH 8.5 of 4.4 x 10(3) M-1 s-1 which is essentially the same as the natural glycosylated form. This recombination rate constant increases in the mutants in the order WT < H42R < H42L << R38L. The value for R38L (5 x 10(6) M-1 s-1) is increased by 3 orders of magnitude relative to the wild-type and is similar to that for human hemoglobin [Mims et al. (1983) J. Biol. Chem. 258, 14219-14232]. Cyanide recombination with the wild-type ferrous form at room temperature is biphasic at pH 6.5 but becomes more monophasic at pH 8.5, again similar to the behavior of the natural glycosylated form, although the Fe(2+)-cyano form of the recombinant enzyme appears to be more unstable at high pH. None of the mutant forms were able to bind cyanide in the ferrous state to any significant extent (Kdiss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (Kdiss approximately equal to 1 mM at pH 7). This behavior contrasts with that of the oxidized forms of the mutants where increases in cyanide dissociation constants are smaller ( < 25 times).(ABSTRACT TRUNCATED AT 250 WORDS)
Meunier
B
B
Glynn Research Institute, Bodmin, Cornwall, United Kingdom.
Rodriguez-Lopez
J N
JN
Smith
A T
AT
Thorneley
R N
RN
Rich
P R
PR
D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002248:Carbon Monoxide / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007834:Lasers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010782:Photolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis
D003486:Cyanides; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D001120:Arginine; D006735:Horseradish Peroxidase