Invited Talk 21st International Conference on Biological Inorganic Chemistry 2025

Electron paramagnetic resonance and X-ray crystallography revealed the unique cold adaptation mechanism in di-Mn enzyme (122307)

Masaki Horitani 1 2
  1. Department of Applied Biochemistry and Food Science, Saga University, Saga, SAGA, Japan
  2. The United Graduate School of Agricultural Science, Kagoshima University, Kagoshima, Japan

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis reaction of inorganic pyrophosphate to two phosphates and plays an essential role for all living organisms. Family II PPase is required Mg and divalent transition metals such as Mn2+ and Co2+ ion for the maximum activity. We previously have succeeded in the purification of PPase from Shewanella sp. AS-11 (Sh-PPase), lives in Antarctic Ocean[1,2]. We utilized electron paramagnetic resonance (EPR) spectroscopy to investigate for solution of Mn-activated Sh-PPase. EPR analyses were confirmed that the active site consists of di-Mn2+ center in Sh-PPase. Di-Mn2+ site yields total spin S = 0 system in the ground state, which is EPR inactive, and has S = 1, 2…5 as second, third… exited states, which are all EPR active and whose splitting are 2J, 4J… higher than the ground state whose J is Heisenberg exchange coupling constant. Our EPR analysis determined the isotropic exchange coupling constant, J = -0.85 cm-1 and axial zero-field splitting (ZFS) parameters, D = ±0.25 cm-1 and -0.058 cm-1 for S = 1 and S = 2, respectively. The axial ZFS for S = 2 and Mn-Mn distance was known to have an empirically linear correlation[3], and this gave Mn-Mn distance 3.57 Å for Sh-PPase. Crystallographic studies revealed a bridged water placed at a distance from the di-Mn centre in Mn-Sh-PPase without substrate. And the water came closer to the metal centre when the substrate was bound. EPR analysis of Mn-Sh-PPase without substrate revealed considerably weak exchange coupling, whose magnitude was increased by binding of substrate analogues. The EPR data indicates that the bridged water has weak bonds with the di-Mn centre, which suggests a ‘loose’ structure, whereas it comes closer to di-Mn centre by substrate binding, which suggests a ‘well-tuned’ structure for catalysis. Thus, we propose that Sh-PPase has loosed structure in absent of substrate and changes the tight structure for efficient catalysis by bound of substrate. I will present about our detailed analysis and proposal of enzymatic reaction mechanism for Sh-PPase.

  1. E. L. Ginting, S. Iwasaki, C. Maeganeku, H. Motoshima, K. Watanabe, Prep Biochem Biotechnol 2014, 44, 480-92.
  2. E. L. Ginting, C. Maeganeku, H. Motoshima, K. Watanabe, Asian Journal of Chemistry 2014, 26, 611-616.
  3. S. V. Khangulov, P. J. Pessiki, V. V. Barynin, D. E. Ash, G. C. Dismukes, Biochemistry 1995, 34, 2015–2025.