Dependence of NO Recombination Dynamics in Horse Myoglobin on Solution Glycerol Content

Abstract

The recombination dynamics of NO with horse heart myoglobin (Mb) following photolysis with a 570 nm excitation pulse were measured by time-resolved absorption with 250 fs temporal resolution. These measurements were carried out in room-temperature solutions in which the glycerol concentration was varied from 0 to 90% (w/v). The recombination of NO is nonsingle exponential in all cases, but becomes faster as the glycerol concentration is increased. The interpretation of these results is aided by a maximum entropy analysis to determine a distribution of rate processes consistent with the data. This analysis suggests that in buffer there are two dominant rate processes for NO recombination on the subnanosecond time scale, one at ≈10 ps and one at ≈200 ps. The dominant effect of increasing glycerol content is to increase the amplitude of the fast process, with no corresponding significant change in rate, and to decrease the amplitude of the slow process, but with a corresponding increase in rate. These results are consistent with a photodissociation process in which photolyzed NO partitions immediately between two distinct populations, one of which has a rapid and the other of which has a slower recombination. The rate of the rapid recombination is independent of glycerol concentration, and therefore decoupled from any protein relaxation process influenced by glycerol, while that of the slower recombination increases at higher glycerol concentration. Further, the partitioning between these two populations also depends on glycerol content, with the relative amplitude of the faster recombination process increasing as the glycerol content is increased. Interpretation of these observations in terms of ligand trajectories following photodissociation, and possible connections of these results with both infrared and crystallographic studies of photodissociated MbCO are discussed.

Spin-dependent mechanism for diatomic ligand binding to heme

“…Protein relaxation slows as well and both protein relaxation and geminate CO recombination become nonexponential (10,16,(24)(25)(26). However, the average time constant for the intrinsic geminate CO recombination process remains close to 1 s.NO recombination is biphasic and occurs with time constants of NO Ϸ 10 ps and NO Ϸ 200 ps under ambient conditions (3,14,15,27). The bimolecular yield for NO is small ⌽bi Ͻ Ϸ0.01.…”

“…In the present study, the ground state potential energy surfaces for the potential energy surfaces of all possible spin states are calculated by using density functional theory (DFT) and compared within the context of a Landau-Zener (L-Z) model for the intrinsic recombination process of each of the ligands. The observed recombination kinetics of CO, NO, and O 2 can be influenced by protein dynamics if the intrinsic recombination rate constant is slower than these dynamics (10,(13)(14)(15)(16). When a combination of temperature and viscosity dependence is used, the general time scale for the intrinsic rate constants can be extracted, and it is these rate constants that are compared in the present work.…”

“…NO recombination is biphasic and occurs with time constants of NO Ϸ 10 ps and NO Ϸ 200 ps under ambient conditions (3,14,15,27). The bimolecular yield for NO is small ⌽bi Ͻ Ϸ0.01.…”

“…The time constant for the rapid phase of NO recombination ( NO Ϸ 10 ps) is comparable to the most rapid phase of protein relaxation at a viscosity of 1 cP (8,28). Two models for the origin of the biexponential kinetics can be divided into a distal effect; two different potential energy wells within the distal pocket of the protein, (13,15,29) and a proxmial effect; a time-dependent barrier caused by iron out-of-plane motion (14,30). As the solvent viscosity is increased, NO recombination becomes increasingly single exponential with a time constant of NO Ϸ10 ps (15).…”

“…Two models for the origin of the biexponential kinetics can be divided into a distal effect; two different potential energy wells within the distal pocket of the protein, (13,15,29) and a proxmial effect; a time-dependent barrier caused by iron out-of-plane motion (14,30). As the solvent viscosity is increased, NO recombination becomes increasingly single exponential with a time constant of NO Ϸ10 ps (15). Because solvent relaxation slows with increasing viscosity, it follows that NO recombination occurs more rapidly than protein relaxation at high solvent viscosity.…”