X-ray Crystallography of Redox Active Proteins

Mark Wilson

June 16th, 2011

X-ray Crystallography Basics

Optimistic workflow for crystallography Experiment Schematic

http://en.wikipedia.org/wiki/X-ray_crystallography

Fourier TransformFourier Transform-1

Monochromatic X-rays typically used

Common Crystallographic Terminology

Resolution: Measured in Ångstroms; “higher” resolution corresponds to smaller number

Unit cell: Basic building block of crystal; can generate the entire crystal by translation

Asymmetric unit: Most fundamental unit of crystal; must be rotated/translated to Asymmetric unit: Most fundamental unit of crystal; must be rotated/translated to make the unit cell

Space group: collection of symmetry operations that build the unit cell from the asymmetric unit

Rsym/Rmerge: A measure of data quality; fractional disagreement between “identical” measurements

R/Rfree: A measure of model quality; fractional disagreement between model and data

Structures Are Models of Electron Density

Calculate density

Diffraction data (one of ~360 images) Electron density with model

Calculate density

Build model

Traditional X-ray Sources: Rotating Anodes

• Produce X-rays by bombarding a metal anode with high energy electrons

http://en.wikipedia.org/wiki/X-ray_tube

C: cathode W: window A: anode T: targetR: rotorS: stator

electrons

• Produced X-rays have a fixed energy that depends on anode metal

• Rotation increases X-ray flux by dissipating heat

Modern X-ray Sources: Synchrotrons

ESRF; Grenoble, France

Confined electron beams moving in circular orbits at nearly the speed of light produce polychromatic X-rays

Synchrotron radiation

http://en.wikipedia.org/wiki/Synchrotron_light_source

Synchrotron storage rings produce bright, tunable X-rays that allow data to be collected from difficult samples

Intense X-rays require cryocooled samples to limit radiation damage

Future X-ray Sources: Free Electron Lasers

FEL design: A “straight” synchrotron Single particle imaging with FEL light

http://hasylab.desy.de/facilities/sr_and_fel_basics/fel_basics/index_eng.htm

Produce femtosecond pulses of X-rays so intense that it converts sample to plasma

Will be able to measure diffraction from single molecules, eliminating need for crystals

Photoelectron Generation and Radiation Damage

Damage to crystal by X-ray beam Photoelectron trajectory and energy

Beam center

http://biop.ox.ac.uk/www/garman/projects.html

Radiation damage is sample, wavelength, dose, and temperature-dependent

14.4 KeV 17.8 KeV

Sanishvilli et al., PNAS, 108 (5), 6127

Redox Proteins Present Challenges for X-ray Crystallography

• Crystals illuminated with X-rays are highly reducing environments

• Some ongoing studies on including radical quenchers in buffers

Issues Current Solutions

• Redox-active groups are typically more sensitive to radiation damage (e.g. disulfides, cofactors, etc.)

• Metals absorb X-rays well, generate damage, and can themselves be reduced

• Minimize time, dose, temperature, wavelength(?)

• Avoid elemental absorption edges when choosing wavelength

Note: Neutron diffraction suffers from none of these problems

Case Study: Dicamba MonooxygenaseCase Study: Dicamba Monooxygenase

Dicamba Monooxygenase is a Rieske Non-heme Iron Oxygenase (RO)

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is an herbicide that is rapidly degraded by soil microbes

Dicamba demethylase is a three component RO responsible for the first step in dicamba degradation

ROs: Functionally Plastic Prokaryotic Oxygenases

Rieske Cluster Redox scheme for three component ROs

Imbeault N Y R et al. J. Biol. Chem. 2000;275:12430-12437

In total, ROs receive two electrons from two NADH; Resting Fe2+ state of non-heme iron

Unresolved Questions about ROs

What is the active oxidant at the non-heme iron: peroxide, oxy radical, or high valency iron-oxo species?

What are the structural determinants that direct oxygen addition to substrate?

Can ROs be engineered or selected to degrade particular pollutants?

Dicamba Monooxygenase (DMO) is the First Structurally Characterized Rieske

Demethylase

Proposed reaction scheme for DMO

Dumitru et al., JMB, 392(2), 2009

DMO catalyzes insertion of oxygen into a C-H bond: difficult and similar to cytochrome p450

DMO is a Obligate Trimer

Monomeric subunit

Trimer

Dumitru et al., JMB, 392(2), 2009

Electron Flow Within DMO-Connecting the Rieske and Non-heme Iron Sites

Surprisingly, the Rieske-mononuclear iron electron transfer path is still ambiguous in ROs

Dumitru et al., JMB, 392(2), 2009

Substrate Binding in DMO is Atypically Selective

Dumitru et al., JMB, 392(2), 2009

DMO appears to be highly selective for its xenobiotic substrate

DMO is Bound to Dioxygen in the Crystal

Crystals dramatically bleached in X-ray beam, indicating photoreduction

Can this be physiological in the

1.75 Å; 2Fo-Fc 1.2σ (blue), Fo-Fc 4σ (green)

Dumitru et al., JMB, 392(2), 2009

Can this be physiological in the free enzyme?

Product is Displaced in DMO Active Site After Demethylation

Dumitru et al., JMB, 392(2), 2009

Suggests means for ejecting product after catalysis

Demethylation is Strongly Selected by DMO Active Site

Other structurally characterized ROs are

Dumitru et al., JMB, 392(2), 2009

characterized ROs are aromatic oxygenases

Oxidant Possibilities in ROs

Both schemes are consistent with chemistry catalyzed by DMO, but peroxo oxidant is not

Summary

Intense modern synchrotron X-ray sources provide ample opportunity for radiation damage

Redox active proteins are particularly vulnerable to radiation damage

Special precautions can limit, but not prevent, X-ray induced redox changesSpecial precautions can limit, but not prevent, X-ray induced redox changes

Dicamba monooxygenase is a atypical RO that specifically demethylates an herbicide

DMO catalyzes oxygen insertion into a C-H bond, which is chemically challenging

Due to complications from the Rieske cluster, less in known about the active oxidant in ROs than in other oxygenases

Acknowledgements

DMO Project

Dr. Razvan DumitruDr. Wen JiangDr. Donald Weeks

Current lab

Dr. Jiusheng LinPeter Madzelan Nicole Milkovic Janani PrahladJanani Prahlad

Funding: NIH, RBC

Data Collection

APS, BioCARS 14BMC