Mo(V) co-ordination in the periplasmic nitrate reductase from Paracoccus pantotrophus probed by electron nuclear double resonance (ENDOR) spectroscopy.
The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus. Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g [resting] increases. This signal is split by two interacting protons that are not solvent-exchangeable. X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V). Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH(-) or H(2)O groups. One of these signals was also lost when the enzyme was redox-cycled in the presence of azide. Since these protons are very weakly coupled OH/H(2)O groups, they are not likely to be ligated directly to the Mo(V). This suggests that protonation of a Mo(VI)zO group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV). A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22-24 MHz. The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo-proton distance of approx. 3.2 A, consistent with this being one of the beta-methylene protons of a Mo-Cys ligand.
Butler
Clive S
CS
School of Biological Sciences, Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich NR4 7TJ, UK.
Fairhurst
Shirley A
SA
Ferguson
Stuart J
SJ
Thomson
Andrew J
AJ
Berks
Ben C
BC
Richardson
David J
DJ
Lowe
David J
DJ
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D006150:Guanine Nucleotides / Q000737:chemistry; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000737:chemistry*; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry*; D009566:Nitrates / Q000737:chemistry; D010230:Paracoccus / Q000201:enzymology*; D011622:Pterins / Q000737:chemistry; D016415:Sequence Alignment
D006150:Guanine Nucleotides; D009566:Nitrates; D011622:Pterins; C063761:molybdopterin guanine dinucleotide; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase