V-51 Solid-State NMR and Density Functional Theory Studies of Vanadium Haloperoxidases
EMSL Project ID
30492
Abstract
This is an application in response to the proposal call "Science Theme: Biological Interactions and Dynamics". We request standard, general non-proprietary access to the 21.1 T (900 MHz) solid-state NMR instrument at EMSL. We additionally request access to the Molecular Science Computing facility to pursue Density Functional Theory calculations of the vanadium haloperoxidase active site models. We will acquire 51V solid-state spectra of vanadium chloroperoxidase active site mutants. We will in parallel use computational facilities to calculate NMR parameters of the active site models of vanadium haloperoxidases. Vanadium haloperoxidases are universally present in marine microalgae, terrestrial fungi, and some lichens and are thought to be involved in the biosynthesis of halogenated natural products. Despite many studies of haloperoxidases encompassing their structure and function the mechanism of their substrate specificity remains controversial because vanadium in these enzymes is diamagnetic "spectroscopically silent" V(V) hampering direct analysis of the vanadate cofactor. We have recently demonstrated that 51V solid-state NMR spectroscopy in combination with Density Functional Theory can be employed as the direct probe of the vanadium cofactor in haloperoxidases, and the spectra are exquisitely sensitive to the coordination environment, including the protonation states of the vanadate cofactor. Our recent successful visit to use the 900 MHz NMR instrument at EMSL enabled us to acquire field-dependent 51V SSNMR spectra of vanadium chloroperoxidase and its R360A active site mutant. The spectra revealed the first experimental evidence of the pH-dependence of the protonation environment of vanadate. We propose to take this work to the next level to examine the 51V spectra of additional active site mutants of vanadium chloroperoxidase, as well as the peroxo intermediates of the wild type and the mutant proteins. We will in parallel perform Density Functional Theory calculations of the NMR parameters using the extended active site models of vanadium chloroperoxidase.
The use of the 21.1 T NMR spectrometer is necessary for this work as we have discovered that the peroxo intermediates as well as the highly protonated states of the resting form of the vanadate cofactor in vanadium haloperoxidases have large quadrupolar coupling constants, and the quality of the data is diminished at lower fields. Furthermore, when available, the field-dependent spectra allow for more precise estimates of the NMR parameters, as we have demonstrated in our most recent work. The availability of computational time for the DFT calculations will be critical because we do not have at the moment sufficient computing power in Delaware to examine extended active site models.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-08-18
End Date
2009-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members