biophysics of metalloenzymes

Biophysics of Metalloenzymes

Topics and Themes:

1) (Metallo-) Proteins and Enzymes in the Cell

2) Some Principles of Coordination Chemistry

3) Methods for Investigation at Molecular Level

4) Overview on Metal Cofactors in Biology

5) Cofactor Assembly and Maturation

6) Excitation-Energy and Electron Transfer

7) Proton Transfer

8) Metal centers in Photosynthesis and Water Oxidation

9) Biological Hydrogen Catalysis

10) Metal Cofactors in Nitrogen Fixation

11) Carbon Oxide Conversion at Metal Sites

12) Molybdenum Enzymes

13) Oxygen Reactions

14) Metal Centers in Human Diseases

15) Bioinspired Materials

Nitrogenase protein x107

x108

Fe3O4 catalyst (ferrit)

300 bar

500 °C

~20 % Ausbeute

Turnover 10 s-1 => 150 Mt / 50t(enzyme) year

Ammonia-plant (Haber-Bosch) x10-3

150 Mt / year

Nitrogen for Industry

Prof. Gerhard Ertl (78)

Fritz-Haber Institut Berlin

Chemie Nobelpreis 2007

Fritz Haber Nobelpreis 1918, Carl Bosch Nobelpreis 1931

Detailed mechanism of N2 catalysis at catalyst surface

Ertl hat die Grundlage der modernen Oberflächenchemie geschaffen, die so unterschiedlichen Vorgängen wie

dem Rosten von Eisen oder der Wirkung eines Katalysators im Auto nachgeht. Ertl wurde 1936 in Stuttgart

geboren. Er promovierte 1965 in physikalischer Chemie an der Technischen Universität München und arbeitete

nach verschiedenen Stationen - auch in den USA - seit 1986 am Fritz-Haber-Institut in Berlin.

Haber-Bosch Process

ca. 1.5 % of world energy demand !

http://www.wissenschaft-online.de/artikel/907579

Hydrogen from N2 Fixation

http://esraa-chemist.blogspot.de/2010/12/biohydrogen-produced-in-air.html

N2 Synthetic Chemistry

Arashiba et al. Nature Chemistry: 3, 120–125 (2011)

Low TON and TOF !

N2 binding energy 945 kJ/mol

Biological Nitrogen Cycle

http://en.wikipedia.org/wiki/Nitrogen_fixation#mediaviewer/File:Nitrogen_Cycle.svg

N2-fixing Organisms

Free-living bacteria: Diazotrophs are cyanobacteria, e.g. trichodesmium, green

sulfur bacteria, azotobacteraceae, rhizobia, and Frankia, e.g. in soil.

Plants that contribute to nitrogen fixation include the legume family – Fabaceae –

with taxa such as beans, lupines, and peanuts. They contain symbiotic bacteria

called Rhizobia within nodules in their root systems.

Others: of 122 genera in the Rosaceae, only 4 are capable of fixing nitrogen.

Root nodules with billions of N2-fixing bacteria (Knöllchenbakterien)

Nitrogenase

Crystal structure (1 Å)

Oliver Einsle (Göttingen)

Organisation

Genes for Nitrogenase

Oldroyd, Current Opinion in Biotechnology 2014, 26:19–24

Biophysics of Metalloenzymes M. Haumann SS2014

Overall Reaction Cycle

Metal Cofactors

P-cluster 8Fe7S

FeMo cofactor (M-cluster)

1Mo7Fe9S1C

Substrates

FeMoco

Val70

Mo

substrate

Mutagenesis broadens substrate specificity

(Markus Ribbe, UC-Irvine)


What is X?

x

Spatzal et a. Science

2011; 334, 940.

Crystallography:

X is carbon, C

Fig. 3. (A) Comparison of the calculated V2C

XES spectra of FeMoco with an interstitial C4–

(black), N3– (blue), and O2– (red) and of the

spectra of the P clusters (gray). (B) Calculated

V2C XES spectra of FeMoco with an interstitial

C4– (black) and the P clusters (gray). (C)

Experimental difference spectrum of FeMoco

with the P clusters (gray), as well as calculated

difference spectra of the P clusters with FeMoco

containing interstitial C4– (black), N3– (blue), and

O2– (red).

What is X ff

Lancaster et al. Science 334, 974 (2011)

X-ray emission spectroscopy

XES: X is carbon, C


P-cluster Assembly

Fig. 4. Stepwise assembly of P-clusters in NifDK (A) and EPR features of the assembly intermediates in the dithionite-reduced (B)

and IDS-oxidized (C) states. (A) The different conformations of P-cluster during assembly are represented by ΔnifH NifDK (left),

which contains two [Fe4S4] cluster pairs (or P*-clusters); ΔnifBΔnifZ NifDK (middle), which contains one P-cluster and one [Fe4S4]

cluster pair (or P*-cluster); and ΔnifB NifDK (right), which contains two P-clusters. Maturation of the “first” P-cluster requires NifH,

whereas maturation of the “second” P-cluster requires both NifH and NifZ. Formation of the P-cluster at the α/β-subunit interface also

induces a conformational change of the α-subunit, which “opens” up the M-cluster site. (B and C) The P*-cluster in ΔnifH NifDK (B,

left) displays a characteristic S=1/2 signal at g=2.05, 1.93, and 1.90 in the dithionite-reduced state; the P-cluster in ΔnifB NifDK (C,

right) displays a characteristic g=11.8 parallel-mode signal in the IDS-oxidized state; the ΔnifBΔnifZ NifDK (B and C, middle) displays

both P*- and P-specific signals at ~50% intensity.

Hu & Ribbe,

Biochimica et

Biophysica Acta 1827

(2013) 1112–1122

Markus Ribbe, UC

Irvine

Hu & Ribbe, J Biol Chem 288, 13173–13177, 2013

Formation of an 8FeC core

M-cluster Assembly

C-atom insertion

Mo Exchange


Cluster Transfer


Key AS residues

FeMo protein

Energetic Bottleneck

Current Opinion in Chemical Biology 2006, 10:101–108

Putative Intermediates

Barney et al. Dalton Trans. 2006, 2277-2284

Rate Constants

Duval, Proc Natl Acad Sci U S A 2013, 110(41):16414-9

Protein association

Conformational gating of ET

Interplay of ET/PT and

Chemistry

PCET ?

Gating of ET

Where does Hydride Bind?

Hoffman, Acc Chem Res 2013


N2 Mechanism

Seefeldt, Annu. Rev. Biochem. 2009. 78:701–22

distal

mechanism

alternating

mechanism

still speculative

Pathways from DFT

Summary

Global nitrogen cycle

Biological N2 fixation

Haber Bosch Process

H2 from N2

Nitrogenase

Crystal structure

Genes

Reaction cycle

Cofactors, P- and M-clusters

Nature of X in FeMoco

Cluster assembly

Intermediates

Reaction mechanism

Literature

Einsle, Nitrogenase FeMo cofactor: an atomic structure in three simple

Steps. J Biol Inorg Chem 2014

Rees, Structural basis of biological nitrogen fixation, Phil. Trans. R. Soc. A 2005 363,

2005

Hoffman, Nitrogenase: A Draft Mechanism, Acc Chem, Res 46, 587–595, 2013

Lancaster, X-ray Emission Spectroscopy Evidences a Central Carbon in the Nitrogenase

Iron-Molybdenum Cofactor, Science 334, 974, 2011

Peters, Exploring new frontiers of nitrogenase structure and mechanism, Current

Opinion in Chemical Biology 2006, 10:101–108

Hu & Ribbe, Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase, J. Biol.

Chem. 2013, 288:13173-13177

Hu & Ribbe, Nitrogenase assembly, Biochimica et Biophysica Acta 1827 (2013) 1112–

1122

Seefeldt, Electron transfer in nitrogenase catalysis, Current Opinion in Chemical Biology

2012, 16:19–25

biophysics of metalloenzymes

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