Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin

S. Franzen (1) , Audrius Jasaitis (1) , J. Belyea (1) , S. Brewer (1) , Romain Casey (1) , A. Macfarlane (1) , Jean-Louis Martin (1, 2, 3) , R. Stanley (1) , Marten H. Vos (1)

1 Department of Chemistry
2 LOB - Laboratoire d'optique et biosciences
3 Temple Univ, Dept Chem, Philadelphia, PA 19122 USA

The recombination dynamics of NO with dehaloperoxidase ( DHP) from Amphitrite ornata following photolysis were measured by femtosecond time- resolved absorption spectroscopy. Singular value decomposition ( SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximate to 9 ps and tau(12) approximate to 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S) 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site > 3 angstrom from the heme iron with the greater barrier corresponding to a S) 5/ 2 spin state. The second SVD basis spectrum represents a time- dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximate to 3 ps and tau(22) approximate to 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP- NO and horse heart myoglobin ( HHMbNO) or sperm whale myoglobin ( SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild- type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP- NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP- NO also hold for the H64V mutant of SWMb ( H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% ( v/ v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild- type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.

S. Franzen, Audrius Jasaitis, J. Belyea, S. Brewer, Romain Casey, et al.. Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin. Journal of Physical Chemistry B, 2006, 110 (29), pp.14483. ⟨10.1021/jp056790m⟩. ⟨hal-00824480⟩