PDBsum entry 3cus

Oxidoreductase

PDB id: 3cus

Contents

Protein chains

262 a.a. *

544 a.a. *

Ligands

SF4 ×6

F3S ×3

GOL ×5

FCO ×3

Metals

_NI ×3

_MG ×3

Waters ×1013

* Residue conservation analysis

PDB id:

3cus

Name:

Oxidoreductase

Title:

Structure of a double ile/phe mutant of ni-fe hydrogenase refined at 2.2 angstrom resolution

Structure:

Periplasmic [nife] hydrogenase small subunit. Chain: a, b, c. Synonym: nife hydrogenlyase small chain. Engineered: yes. Periplasmic [nife] hydrogenase large subunit. Chain: q, r, s. Synonym: nife hydrogenlyase large chain. Engineered: yes. Mutation: yes

Source:

Desulfovibrio fructosovorans. Organism_taxid: 878. Strain: wild type. Gene: hyda. Expressed in: desulfovibrio fructosovorans. Expression_system_taxid: 878. Gene: hydb. Expression_system_taxid: 878

Resolution:

2.20Å R-factor: 0.187 R-free: 0.222

Authors:

A.Volbeda

Key ref:

F.Leroux et al. (2008). Experimental approaches to kinetics of gas diffusion in hydrogenase. Proc Natl Acad Sci U S A, 105, 11188-11193. PubMed id: 18685111 DOI: 10.1073/pnas.0803689105

Date:

17-Apr-08 Release date: 05-Aug-08

Protein chains

P18187  (PHNS_SOLFR) -  Periplasmic [NiFe] hydrogenase small subunit from Solidesulfovibrio fructosivorans

Seq: 314 a.a.

Struc: 262 a.a.

Protein chains

P18188  (PHNL_SOLFR) -  Periplasmic [NiFe] hydrogenase large subunit from Solidesulfovibrio fructosivorans

Seq: 564 a.a.

Struc: 544 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, Q, B, R, C, S: E.C.1.12.2.1 - cytochrome-c3 hydrogenase.
• Reaction: 2 Fe(III)-[cytochrome c3] + H2 = 2 Fe(II)-[cytochrome c3] + 2 H⁺
• Cofactor: Iron-sulfur; Ni(2+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1073/pnas.0803689105

Proc Natl Acad Sci U S A 105:11188-11193 (2008)

PubMed id: 18685111

Experimental approaches to kinetics of gas diffusion in hydrogenase.

F.Leroux, S.Dementin, B.Burlat, L.Cournac, A.Volbeda, S.Champ, L.Martin, B.Guigliarelli, P.Bertrand, J.Fontecilla-Camps, M.Rousset, C.Léger.

ABSTRACT

Hydrogenases, which catalyze H(2) to H(+) conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H(2) toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.

Selected figure(s)

Figure 1.
Structural models of the three enzymes. A gives an overview of the tunnel network; B is a closeup of the tunnel near the active site in the WT. C, D, and E are closeups of the MM and FI mutants, as indicated. In C, an arrow points to the second conformation of M122. A conserved hydrophilic cavity is shown in blue in E.

Figure 3.
Comparison of the kinetics of CO inhibition of H[2] oxidation in PFV experiments (26). The current i has been normalized by its value i(0), measured before CO was added. Left shows the short-term change in current, whereas the end of the relaxation is shown on Right. The dimensionless volumic fractions of solutions saturated under 1 atm of CO at 25°C and injected at time 0 (see SI Text) were x = 7 × 10^−3 (A, WT), 12 × 10^−3 (B, FI), 2.5 × 10^−3 (C, MM). Electrode rotation rate 2 krpm, pH 7, T as indicated. The fits of the data to Eq. 1 in SI Text are shown as dashed black lines.

Figures were selected by the author.