Hydrophobic Distal Pocket Affects NO−Heme Geminate Recombination Dynamics in Dehaloperoxidase and H64V Myoglobin

Abstract

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) approximately 9 ps and tau(12) approximately 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 A 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) approximately 3 ps and tau(22) approximately 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.

Ligand Dynamics and Early Signaling Events in the Heme Domain of the Sensor Protein Dos from Escherichia coli

“…This kinetics does not alter significantly when 60% glycerol is present (not shown). This observation is consistent with previous observations on other heme proteins that the time constant of the fastest phase of geminate NO recombination is independent of glycerol concentration (32,33) and supports the idea that these phases reflect predominantly barrierless rebinding.…”

“…5C). With increasing viscosity, the overall rebinding becomes faster, which is generally consistent with previous studies on NO rebinding in other heme proteins (32,33), with CO rebinding to protoheme (34,35) and with a barrier for CO escape in DosH as deduced from the temperature-dependence studies (Fig. 5B).…”

“…In previous studies on the effect of glycerol on biexponential picosecond NO geminate rebinding with the globin proteins Mb (33) and dehaloperoxidase (32) an increase in the amplitude (but not the rate) of the fast phase of rebinding was observed, but in these cases it was associated with an increase in the rate (on the (10 2 ps) Ϫ1 scale) of the slow phase. These results were interpreted in terms of immediate population of two dissociated NO rotamers, only one of which would directly rebind.…”

Distal histidine conformational flexibility in dehaloperoxidase fromAmphitrite ornata

“…The integrity of the protein was analyzed by assessing its enzymatic activity toward 2,4,6-tribromophenol (TBP), a natural substrate of DHP (Belyea et al, 2005;Franzen et al, 2006). Crystals were grown by the hanging-drop vapor-diffusion method from 0.2 M ammonium sulfate and 26-34% polyethylene glycol 4000 at 277 K. The starting protein concentration was 8 mg ml À1 .…”

Applications of X-Ray Transient Absorption Spectroscopy in Photocatalysis for Hydrogen Generation

“…On the basis of the time evolution of characteristic optical features for different intermediate states, optical transient absorption (OTA) (or emission, OTE) spectroscopy tracks populations and energetics, as well as coherence and correlation among different transient species in photochemical reactions. With the exception of time-resolved vibrational spectroscopy, which indirectly measures transient structures [22][23][24], optical transient spectroscopy provides no direct structural information for reaction intermediates. These transient structures are often inferred by theoretical calculations without experimental verification.…”