M.Sc. Project IntroductionNitrogen-fixing Enzymes

M.Sc. Candidate: Egill SkulasonSupervisor: Hannes Jonsson

Co-supervisor: Magnus Mar KristjanssonRaunvisindastofnun Haskola Islands

Efnafraedistofavklubbur

31st of July 2003

Contents

• Introduction• Background• Nitrogenase• Methodology• FeMo Cofactor

– Model Structures– Reactivity

• Biological Ammonia Synthesis• Summary and Future Study

Introduction

• Project Goal: Study the biological ammonia synthesisusing density functional calculations (DFT) on models ofthe active center of nitrogenase.

• Nitrogenase is a catalytic enzyme found in many bacteria,and is central to biological ammonia synthesis.

• Understanding of this process may lead to a more gentlemanufacturing of ammonia, lowering pollution and energycosts.

• Background: Density Functional Calculations andModeling of the Biological Ammonia Synthesis by ThomasHolm Rod, Ph. D. (2000) Technical University ofDenmark (DTU) and Center for Atomic-Scale MaterialsPhysics (CAMP).

Background

• The conversion of atmospheric N2 into abiologically accessible form of nitrogen such asammonia, is termed nitrogen fixation.

• Since the element N is present in manybiomolecules, such as amino acids, nitrogenfixation is a prerequisite for life.

• In spite of the vast quantities of atmospheric N2,the sources of biologically accessible nitrogen arefew.

• Oxidation of N2 to nitrogenoxides by lightning andcombustion.

• The commercial Haber-Boschprocess where N2 reacts withhydrogen on a Fe or Ru basedcatalyst to form ammonia.

• The enzyme catalyzedammonia synthesis where N2reacts with electrons andprotons to form ammonia.

The Main Sources ofBiologically Accessible Nitrogen

Ref: L. Stryer, Biochemistry, 4. Ed. (W.H. Freeman and Company, New York, 1995), p. 714.

Nitrogenase• Nitrogenase consists of two metalloproteins: Fe protein

and MoFe protein (named after their metal clusters).• The Fe protein contains a ferredoxin (4Fe-4S), which is

known to play a role in electron transfer in many proteins.W. Kaim and B. Schwederski, Bioinorganic Chemistry: Inorganic Elements inthe Chemistry of Life (John Wiley & Sons, New York, 1991).

• The MoFe protein contains two unique cluster pairs,namely the P-cluster and the FeMo cofactor (FeMoco).

• FeMoco is most likely the active center where N2 bindsand is reduced.

Nitrogenase

N. Schindelin et al., Structure of ADP · AlF4- -stabilized nitrogenase complex and its

implications for signal transduction, Nature 387, (1997), PDB ID code: 1N2C.

Ferredoxinreduced e- areacceleratedthrough the P-cluster towardsthe FeMoco.

The Metalloclusters

Sulfur: yellowIron: purpleMolybdenum: blue

N. Schindelin et al., Structure of ADP · AlF4- -stabilized nitrogenase complex and its

implications for signal transduction, Nature 387, (1997), PDB ID code: 1N2C.

The Methodology• Density Functional Calculation (DFT) forms the basis in

this Ph. D. thesis and will be used in this M.Sc. study.• The exchange correlation functionals applied in the Ph. D.

study, by Rod, were the PW91 and the RPBE functionals.The latter will probably be used for current study.

• DACAPO: an DFT calculating program which use plane-wave pseudo-potentials.

Model Structures of theFeMo Cofactor

N2 Adsorption on Models I and II

Adsorption energies: Model I 0.0 eVModel II -0.1 eV

Reactivity of the FeMoco

• H bonding (H+ + e-) and H2 formation• N2 adsorption

– modeling of the interaction with thesurrounding protein

• The energy of a system is always calculatedin relation to model I and the incomingsubstrate, usually in gas phase.

H Binding andH2 Formation of

Model I

To study the effect of the e- andH+ flow to the FeMoco, 1-4 Hatoms where added to model I.

N2 Adsorption

Modeling of the Interaction withthe Surrounding Protein

• There is a possibility that an e- is transferred to thecomplex without a simultaneous transfer of a proton.

• It is clear that the enzyme must have sites close to theactive site that can act as proton donors during nitrogenhydrogenation.

• Such a donor might either be an amino residue or an H2Omolecule.

• They therefore add to their system a weak base, NH4+ or

H3O+, in the vicinity of the cofactor.

N2 Adsorption on Model I with andwithout the Proton Donor NH4

+

Biological Ammonia Synthesis

A Short Summary• In this thesis a simple model of the FeMoco has been

investigated theoretically.• Two different clusters have been studied to mimic the central

part of the FeMoco.• The effect of the surroundings have also been included in a

crude way by invoking proton donors in the vicinity of thecofactor.

• The interaction of the FeMoco with the substrates H, H2 andN2 have been calculated and most of the observations agreewith experimental results. (Not discussed in this presentation).

• A reaction path, of the biological ammonia synthesis, hasbeen calculated.

For Future Study

• Investigate the real environment of thenitrogenase and make new models.

• Simulate the high chemical potential of theelectrons and protons.