Structure-function relationship of sensitive to oxygen

Yvain Nicolet Structure/Function Relationships of Metalloproteins Metalloproteins Unit What I mean by Metalloproteins? Metalloproteins contain transition metals (d-elements) Non-metals

Transition metals

Rare Earth Elements

➣ Different oxidation states. (Redox catalysis, electron transfer...) ➣ Lewis acids. (Non-redox catalysis) ➣ Properties modified by the ligands. (fine-tuned reactions) ➣ New chemistry available.

NiFe- from D. gigas FeFe-hydrogenase from D. desulfuricans Fe-hydrogenase from M. jannaschii

Volbeda et al., Nature 1995 Nicolet et al., Structure 1999 Shima et al., Science 2008

Development of bio-inspired catalysts to use hydrogen as a renewable fuel.

NifDK (MoFe-protein) FeMo- NifH (Fe-protein) NifH (Fe-protein)

Schindelin et al., Nature 1997

N2 is the main source of nitrogen for life. Nitrogenase is the enzyme responsible for its conversion into forms useable by plants. dehydrogenase/acetyl-CoA synthase The Wood-Ljungdahl pathway

ACS CODH CODH ACS ACS

Darnault et al., NSB 2003

CO2 fixation, C-C bond formation. Fumarate nitrate reduction regulator (FNR)

Fenton reaction with Fe2+

Volbeda et al., Sci. Adv. 2015

Fine-tuned adaptation to oxidative stress by facultative anaerobes (numbers of pathogens). More metalloproteins

Respiration Photosynthesis Respiratory complex I (Type I NADH dehydrogenase) Photosystem I Stroma Photosystem II

Matrix

Inner membrane PC + Fdx + hv <---> PC + Fdx Lumen red ox ox red

Intermembranar space 2H2O + 4hv + 2PQ ---> O2 + 2PQH2

+ + + + NADH + H +CoQ + 4H in ---> NAD +CoQH2 + 4H out A mineral origin of life? + - 0 FeS + H2S ---> FeS2 + 2H + 2 e E ’=-620 mV Wächtershäuser, Microbiol. Rev. 1988 Pyrite-pulled surface metabolism

Hydrogenase CODH ACS (NiFeS4) (NiFe4S9) (Ni2Fe4S10)

Pyrite (FeS2)

(Fe5NiS8)

Volbeda et al., Coord. Chem. Rev. 2005 Wood-Ljungdahl pathway The Metalloproteins Unit Mechanism Lewis chemistry Structure-function relationships

Radical-based chemistry

Assembly – degradation, regulation

X-ray crystallography Functional analyses Theoretical calculations The Metalloproteins Unit

A unique set of gloveboxes to work under anaerobic conditions “from gene to structure”

A unique and newly installed automated arm to analyze our crystallization plates over time

A LC-MS dedicated to functional analyses

A crystallization robot to screen up to 1248 conditions

A new versatile glovebox to prepare cryo-EM grids Radical S-adenosyl-L- (SAM) enzymes

SAM 5’-dA• 5’-dAH

L-Met L-Met

Ÿ Conserved Cx3Cx2C motif.

Ÿ reductive homolytic cleavage of SAM leading to a highly reactive 5’dAŸ radical species as the initiator of the radical-based reaction. Radical S-adenosyl-L-methionine (SAM) enzymes

Ÿ Catalyze difficult reactions often not accessible with the two-electron based chemistry. Ÿ Over 120 000 unique sequences identified in all three domains of life. Ÿ More than 70 different chemical reactions characterized. Ÿ Found at key steps in the biosynthesis of many cofactors and antibiotics. HydE: a radical SAM enzyme involved in the maturation of the FeFe-hydrogenase active site

Nicolet, et al, JBC. 2008

Pilet, et al, FEBS Lett. 2009 Nicolet, et al, Chembiochem. 2015 ? Pagnier, et al, PNAS. 2016 FeS-cluster degradation upon oxygen exposure Our experimental setup

HydE from T. maritima

Ÿ Cx7Cx2C motif Ÿ Cx3Cx2C motif Ÿ Fe4S4 cluster after in vitro reconstitution Ÿ Fourth ligand is SAM Rubach et al., FEBS Lett. 2005 Ÿ Shielded from solvent when Ÿ Unknown fourth ligand SAM is bound Ÿ Fully exposed to solvent Ÿ Observed as a Fe2S2 cluster in our original structure Nicolet et al., J. Biol. Chem. 2008

Easily reproducible orthorhombic crystals that diffract at up to 1.2 Å resolution In crystallo DTT treatment

Experimental conditions: - Protein purified aerobically - FeS clusters subsequently reconstituted in Experimental conditions: vitro under anaerobic conditions (contains - Same as previously (same drop) 2 Fe4S4 clusters) -Crystal cooling in the glove box (5 mM DTT added - Crystallization under anaerobic conditions in the cryoprotecting solution) (No DTT added) - Crystal cooling in the glove box (No DTT 1.60 Å resolution added)

1.45 Å resolution

DTT A slow decay to reach the apo form

Experimental conditions: Experimental conditions: - Protein purified aerobically - Protein purified aerobically - FeS clusters subsequently reconstituted in vitro - FeS clusters subsequently reconstituted in under anaerobic conditions (contains 2 Fe4S4 clusters) vitro under anaerobic conditions (contains - Crystallization under anaerobic conditions 2 Fe S clusters) 4 4 (5 mM DTT) – leads to a regular Fe S cluster- - Crystallization under anaerobic conditions 2 2 containing protein (5 mM DTT) – leads to a regular Fe S 2 2 - 2 months before flash-cooling in the glove box cluster-containing protein (5 mM DTT) - 1 month before flash-cooling in the glove box 1.35 Å resolution (5 mM DTT)

1.71 Å resolution

With time, the solvent-exposed iron atom is lost. Then the whole cluster is disrupted. Toward a full description of the decay of FeS clusters

Slow

Nicolet et al., PNAS. 2013 Identification of ligands… Rohac, et al, Nature Chem. 2016

A B C

L- + pyruvate L-cysteine + glyoxylate L-cysteine + formaldehyde

D E F

L-cysteine + mercaptopyruvate L-penicillamine + pyruvate L-homocysteine + pyruvate that can act as non-natural substrates

Second crystal frozen after 120min soaking with DTH

Reaction product Se-SAC in the active site of (2R,4R)-MeSeTDA-bound HydE structure HydE at the final freezing time t > 120 min Rohac, et al, Nature Chem. 2016 Kinetics in solution

• Cleavage of the SAM molecule • New Se-C5’ bond formed • Disappearance of the pyruvate moiety replaced by a chloride ion • S-adenosyl-L-selenocysteine (SeAC) density at the end of the reaction • Successful co-crystallization with commercial SAC leads to exactly the same structure In crystallo radical-based chemistry Double difference electron density map

Frozen at t=0 min • t0min ( reference dataset ) Fo0min

Frozen at t=x min • tXmin ( 5 < x < 240 ) FoXmin

ρ double diff map = (|FoXmin| - |Fo0min|)exp(2πiϕcalc 0min)

Red component : ρ0min > ρXmin

Green component : ρ0min < ρXmin

Atoms move from red to green In crystallo radical-based chemistry Dissecting a carbon – sulfur bond formation

In red: what phases out In green: what comes up • C5’-Sδ bond breakage 1.86 Å ➞ 3.92 Å

• Sδ shifts toward Feu 3.25 Å ➞ 2.84 Å

• Formation of a new Se-C5’ bond 3.90 Å ➞ 2.11 Å

• Cleavage of Se-C2 bond and disappearance of the pyruvate moiety 2.00 Å ➞ ∞

• Almost perfect alignment of atoms involved in bond cleavage or formation

Rohac, et al, Nature Chem. 2016 What hapenned to the pyruvate moiety?

Preparation of specifically 13C-labelled substrate 1H-13C-NMR kinetic study of HydE 1 Pyruvate-3-13C

2 (2R,4R)- (2RS,4R)-Me-13C-TDA

(2S,4R)-

3 Kinetic experiment with HydE enzyme

t0 tfinal

Ethanol

At the end, free pyruvate is released from the substrate. Rohac, et al, Nature Chem. 2016 Overall mechanism

Theoretical calculations

Nicolet, et al, PNAS. 2009 Rohac, et al, Nature Chem. 2016 The nosiheptide antibiotic

Zhang et al., NCB 2011

The Radical S-adenosyl-L-methionine (SAM) lyase is responsible for the convertion of L-tryptophan into MIA Postulated mechanism of NosL

Ca-Cb bond Formaldehyde Zhang et al., NCB 2011 cleavage by-product

Indole N-centered radical formation Recombination X-ray structure of NosL from Streptomyces actuosus at 1.8 Å resolution

Structure-based mechanism for the activation of L-tryptophan

Nicolet et al., Angew. Chem. 2014 Radical intermediate trapping – EPR analysis

EPR spectrum: NosL + L-tryptophan + Same spectrum recorder at 80K SAM + dithionite (45 s; 20K)

An unprecedented carboxyl- radical migration

Sicoli et al., Science. 2016 New proposed mechanism

Sicoli et al., Science. 2016

➣ Path I corresponds to an unproductive reaction. ➣ Path II leads to MIA, which will be further processed for nosiheptide biosynthesis. - How does the carboxyl radical (•CO2 ) migration step proceed?

Is the carboxyl migration a concerted reaction (i.e. a one-step reaction)?

or

• - Is the carboxyl migration a two-step reaction (i.e. the CO2 radical a true intermediate)?

How does the protein control this difficult radical-based reaction? - How does the carboxyl radical (•CO2 ) migration step proceed? Structural investigation using analogues of the radical intermediate?

Sicoli et al., Science. 2016

➣ No movement of the surrounding residues. 2.0 Å resolution ➣ the carboxyl moiety occupies the same site – the indole ring is rotating. Good structural model to perform QM/MM calculations - How does the carboxyl radical (•CO2 ) migration step proceed?

QM/MM calculations: direct migration from Ca to C2 is unlikely (no suitable orbital overlap – a high barrier > 50 kcal/mol) A two-step mechanism is thus investigated

● - Radical intermediate Conformational change (arm rotation) crucial CO2 radical is a minimum to weaken Cα-C bond (EPR-detected) Two conformations for the iminoindolinyl radical species

Remarkably, calculations of the hyperfine couplings lead to the species previously detected by EPR ACKNOWLEDGMENTS

SyMMES-INAC-CEA Serge Gambarelli Jean-Marie Mouesca Giuseppe Sicoli

NMR GROUP-IBS Adrien Favier

Structure/Function Relationships of Metalloproteins MICALIS-INRA Metalloproteins Unit Olivier Berteau METALLOPROTEINS UNIT-IBS Alhoshna Benjdia Juan C. Fontecilla-Camps Pauline Ruffie Laura Zeppieri Patricia Amara FUNDINGS Lydie Martin Roman Rohac Adrien Pagnier