Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.
Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.
Howes
B D
BD
Dipartimento di Chimica, Università di Firenze, Via G. Capponi 9, I-50121 Firenze, Italy.
Heering
H A
HA
Roberts
T O
TO
Schneider-Belhadadd
F
F
Smith
A T
AT
Smulevich
G
G
D019943:Amino Acid Substitution; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D013059:Spectrum Analysis, Raman
D006877:Hydroxamic Acids; D007527:Isoenzymes; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid