EVIDENCE FOR A SLOW TERTIARY RELAXATION IN THE REACTION OF TERT-BUTYLISOCYANIDE WITH HORSERADISH-PEROXIDASE

The kinetics of tert-butyl isocyanide binding to the heme protein hors eradish peroxidase (HRP) at 22 degrees C was examined on all time scal es, from minutes to picoseconds, in aqueous berate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M(-1) s(-1), while in Mb it is 4 x 10(3) M(-1) s(-1). Surprisingly, in view of that difference, pi cosecond and nanosecond photolyses reveal that once the ligand has rea ched the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the cont act pair) is close to 6 x 10(10) s(-1) in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair ar e also similar: for Mb, 10 x 10(10) s(-1), and for HRP, 8.5 x 10(10) s (-1). The rate of rebinding from the protein-separated cage is near 4 x 10(6) s(-1) in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s(-1) in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination n ot seen in mixing experiments. This bimolecular rate constant varies s lightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10( 4) M(-1) s(-1) in two cases, both of which are much faster than is obs erved in mixing experiments, namely, 0.04 M(-1) s(-1). In Mb, photolys is and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombinatio n. Similar measurements for the smaller ligand methyl isocyanide revea led no anomalous behavior. The interpretation proposed involves tertia ry relaxation after ligand escape, which is significant in blocking th e return of the large t-BuNC, but has no apparent effect on smaller li gands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merel y hinted at in earlier work involving the T-state binding kinetics of hemoglobin.