Active Site Structures of the Escherichia coli N-Hydroxylaminopurine Resistance Molybdoenzyme YcbX

Abstract

X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) data have been used to characterize the coordination environment for the catalytic Mo site of Escherichia coli YcbX in two different oxidation states. In the oxidized state, the Mo(VI) ion is coordinated by two terminal oxo ligands, a thiolate S atom from cysteine, and two S donors from the bidentate pyranopterin ene-1,2-dithiolate (pyranopterin dithiolene). Upon reduction, it is the more basic equatorial oxo ligand that is protonated, with a Mo–Oeq bond distance that is best described as either a short Mo4+–OH2 bond or a long Mo4+–OH bond. Mechanistic implications for substrate reduction are discussed in light of these structural details.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism

“…The nature and number of the coordinating donor ligands in wt and C176A NapA have been determined by analyzing the Mo K-edge EXAFS data (Figure ). The Fourier transform of the EXAFS oscillations for oxidized wt NapA (Figure A) clearly shows two peaks that are typical of terminal oxo and thiolate sulfur scatterers in molybdoenzymes and models. − The best fit to the EXAFS data gives a Mo ≡ O bond length of 1.73 Å, an average dithiolene Mo–S bond lengths of 2.44 Å, and four second coordination sphere dithiolene C scatterers at 3.34 Å (Figure A,B). The computational model used for the EXAFS fit is shown in Figure C.…”