The Enzyme Horseradish Peroxidase Is Less Compressible at Higher Pressures

Abstract

Fluorescence line-narrowing (FLN) spectroscopy at 10 K was used to study the effect of high pressure through the prosthetic group in horseradish peroxidase (HRP), which was Mg-mesoporphyrin (MgMP) replacing the heme of the enzyme. The same measurement was performed on MgMP in a solid-state amorphous organic matrix, dimethyl sulfoxide (DMSO). Series of FLN spectra were registered to determine the (0, 0) band shape through the inhomogeneous distribution function (IDF). In the range of 0-2 GPa a red-shift of the IDF was determined, and yielded the isothermal compressibility of MgMP-HRP as 0.066 GPa(-1), which is significantly smaller than that found earlier as 0.106 GPa(-1) by fine-tuning the pressure in the range up to 1.1 MPa. The vibrational frequencies also shifted with pressure increase, as expected. The compressibility in the DMSO matrix was smaller, 0.042 GPa(-1), both when the pressure was applied at room temperature before cooling to 10 K, or at 10 K. At 200 K or above, the bimodal (0, 0) band shape in DMSO showed a population conversion under pressure that was not observed at or below 150 K. A significant atomic rearrangement was estimated from the volume change, 3.3 +/- 0.7 cm(3)/mol upon conversion. The compressibility in proteins and in amorphous solids seems not to significantly depend on the temperature and in the protein it decreases toward higher pressures.

Biology under extreme conditions

“…Under these conditions one does expect the hydration not to play a very prominent role. Nevertheless the compressibilities that are obtained under such conditions are of the same order of magnitude as those obtained at ambient conditions [33]. This points to important contributions from the cavities to the compressibility.…”

High Pressure Processing – a Database of Kinetic Information

“…The inactivation of enzymes by high pressure is related to reversible or irreversible changes of the native structure [1]. The pressure stability of enzymes can vary dramatically ranging from pressure sensitive enzymes (< 400 MPa) such as phosphohexoseisomerase from bovine milk [68] to extreme pressure resistant enzymes (>1 GPa) such as peroxidase from horseradish [69]. However, since there is a structural manifoldness among enzymes catalyzing the same reaction it is not appropriate to categorize enzymes with respect to their pressure (and/or thermal) stability.…”

Effects of mutations in the helix G region of horseradish peroxidase

“…The stability of HRP has a crucial bearing on its applicability and is affected by a number of variables including temperature [8,9,10], calcium content [10], glycosylation [11], pressure [12], irradiation [13] and pH [9,14], along with additional factors such as urea [15,16], guanidinium chloride [15,17] and various solvents including dimethyl sulfoxide [18] and ionic liquids [19]. Directed evolution has been used to alter rHRP thermal stability [20] but we have found no reports of HRP stability manipulation via rational, site-directed mutagenesis.…”