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 expres sed in Escherichia coli. These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxid e and cyanide after photolysis of the reduced forms. The recombinant w ild-type ferrous form exhibited monophasic recombination of carbon mon oxide 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 glycosyla ted 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-t ype and is similar to that for human hemoglobin [Mims et al. (1983) J. Biol. Chem. 258, 14219-14232]. Cyanide recombination with the wild-ty pe 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 natura l glycosylated form, although the Fe2+-cyano form of the recombinant e nzyme 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 ext ent (K-diss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (K-diss congruen t 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). The results are rationalized in terms of the polarity of the route of access and binding site of carbon monoxide a nd cyanide, and the need for a charge-compensating protonation site fo r the stable binding of the cyanide anion, but not for the binding of the neutral carbon monoxide.