4UCW

Structure of the T18V small subunit mutant of D. fructosovorans NiFe- hydrogenase

Summary for 4UCW

• Entry DOI: 10.2210/pdb4ucw/pdb
• Related: 4UCQ 4UCX
• Descriptor: HYDROGENASE (NIFE) SMALL SUBUNIT HYDA, NICKEL-DEPENDENT HYDROGENASE LARGE SUBUNIT, GLYCEROL, ... (9 entities in total)
• Functional Keywords: oxidoreductase, nife-hydrogenase mutant, unready state
• Biological source: DESULFOVIBRIO FRUCTOSIVORANS; More
• Total number of polymer chains: 6
• Total formula weight: 272752.73

Authors

Abou-Hamdan, A.,Ceccaldi, P.,Lebrette, H.,Guttierez-Sanz, O.,Richaud, P.,Cournac, L.,Guigliarelli, B.,deLacey, A.L.,Leger, C.,Volbeda, A.,Burlat, B.,Dementin, S. (deposition date: 2014-12-04, release date: 2015-02-18, Last modification date: 2024-11-20)

Primary citation

Abou-Hamdan, A.,Ceccaldi, P.,Lebrette, H.,Gutierrez-Sanz, O.,Richaud, P.,Cournac, L.,Guigliarelli, B.,De Lacey, A.L.,Leger, C.,Volbeda, A.,Burlat, B.,Dementin, S.
A Threonine Stabilizes the Nic and Nir Catalytic Intermediates of [Nife]-Hydrogenase.
J.Biol.Chem., 290:8550-, 2015

PubMed Abstract: The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production.

PubMed: 25666617
DOI: 10.1074/JBC.M114.630491

Experimental method

X-RAY DIFFRACTION (2.3 Å)