E. coli
catalase (HPII) wild type and mutant
enzymes (
heme dcis-containing) were examined (i) to study the role of a distal
haem cavity residue, asparagine-201, in high spin
ligand binding and (ii) to compare the differences in this binding between
heme d and
protoheme enzymes such as that from beef liver (BLC). High spin
fluoride complexes were formed by all three HPII catalases examined, wild type (201 asn) and 201gln and 201asp mutants, but with a lower
fluoride affinity than that of BLC. The binding of
fluoride was pH-dependent, indicating that a
proton is bound as well as a
fluoride anion. HPII 201glu and 201 asp mutants showed lower affinities for
fluoride than did wild type, unlike their reactions with
cyanide which are essentially independent of the nature of residue 201. The equilibria and rates of
fluoride and
formate binding to BLC were reexamined. The rates of reaction with
formate were similar to those reported previously. Dissociation rates for
fluoride-
catalase are higher than for
formate suggesting that the latter may be bound differently. High spin complexes between
formate and all three HPII forms showed a substantially higher affinity than that of BLC for HPII wild type and progressively lower affinities for the two mutants. As with
fluoride the reactions were pH-dependent, indicating that a
proton is bound together with the
formate anion (or that undissociated
formic acid is the
ligand). The known structures of the
heme groups and
heme pockets involved are discussed.
Formate may be bound by secondary H-bounds within the
heme pocket in both
heme dcis and
protoheme enzymes. The nature of the
heme pocket and the
heme access channel may be more important than the chemical nature of the prosthetic group in controlling both high spin
ligand interactions and reactions with the substrate
hydrogen peroxide.