Recombination of carbon monoxide to ferrous horseradish peroxidase types A and C.
The recombination of carbon monoxide to isoenzymes A2 and C of horseradish peroxidase (HRP) was studied as a function of temperature (2 to 320 K) and pH (5 to 8.3) with flash photolysis and infrared difference absorption.  At low temperatures three geminate recombination processes are observed.  One of these internal processes, denoted by I*, is exponential in time with a rate coefficient that deviates strongly from an Arrhenius behavior below 100 K, implying phonon-assisted tunneling.  The two other processes, denoted by I, are non-exponential in time and related to different carbonyl isomers, as shown by the infrared difference spectra.  The existence of three internal processes indicates that HRP differs considerably from myoglobin where only one internal process, I, is seen.  Moreover, the internal processes in HRP are faster than process I in myoglobin.  At 300 K, only one recombination process from the solvent is observed and it is very slow (lambda s approximately 1 s-1 at 1 atm CO (1 atm = 101,325 Pa)), much slower than the corresponding association process in myoglobin.  Since process I is fast, but binding from the solvent is slow, the barrier at the heme cannot be responsible for the small association rate.  The infrared absorption difference spectra of the amide I/II bands indicate that photolysis and recombination trigger a two-step structural change.  The slow recombination rate at 300 K can thus be explained by the large Gibbs energy of the conformational transition that is necessary to let CO move into the heme pocket.  The partition coefficient for the CO in the heme pocket and the solvent is extremely small, while bond formation with the heme iron occurs in less than 100 nanoseconds.