Spectroscopic and kinetic studies of Arabidopsis thaliana sulfite oxidase: nature of the redox-active orbital and electronic structure contributions to catalysis.
Plant sulfite oxidase from Arabidopsis thaliana has been characterized both spectroscopically and kinetically. The enzyme is unusual in lacking the heme domain that is present in the otherwise highly homologous enzyme from vertebrate sources. In steady-state assays, the enzyme exhibits a pH maximum of 8.5 and is also found to function as a selenite oxidase. Sulfite at the lowest experimentally feasible concentrations reduces the enzyme within the dead-time of a stopped-flow instrument at 5 degrees C, indicating that the A. thaliana enzyme has a limiting rate constant for reduction, k(red), at least 10 times greater than that of the chicken enzyme (190 s(-1)). The EPR parameters for the high- and low-pH forms of the A. thaliana enzyme have been determined, and the g-values are found to resemble those previously reported for the vertebrate enzymes. Finally, the A. thaliana enzyme has been probed by resonance Raman spectroscopy. A detailed analysis of the vibrational spectrum in the region where Mo=O stretching modes are anticipated to occur has been performed with the help of density functional theory calculations, evaluated in the context of the Raman data. Calculated frequencies obtained for two model systems have been compared to experimental resonance Raman spectra of oxidized A. thaliana sulfite oxidase catalytically cycled in both H2(16)O and H2(18)O. The vibrational frequency shifts observed upon (18)O-labeling of the enzyme are consistent with theoretical models in which either the equatorial oxygen or both equatorial and axial atoms of the dioxomolybdenum center are labeled. Importantly, the vibrational mode description is consistent with the active site possessing geometrically inequivalent oxo ligands and a Mo d(xy) redox-active molecular orbital oriented in the equatorial plane forming a pi-bonding interaction solely with the equatorial oxo, O(eq). Electron occupancy of this Mo=O(eq) pi* redox orbital upon interaction with substrates would effectively labilize the Mo=O(eq) bond, providing the dominant contribution to lowering the activation energy for oxygen atom transfer.
Hemann
Craig
C
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Hood
Brian L
BL
Fulton
Meita
M
Hänsch
Robert
R
Schwarz
Günter
G
Mendel
Ralf R
RR
Kirk
Martin L
ML
Hille
Russ
R
D017360:Arabidopsis / Q000201:enzymology*; D002384:Catalysis; D004583:Electrons; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010084:Oxidation-Reduction; D013059:Spectrum Analysis, Raman; D050876:Sulfite Oxidase / Q000737:chemistry*
D050876:Sulfite Oxidase