Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

Hough, Michael A.; Owen, R.L.

doi:10.1016/j.sbi.2021.07.007

Cited by 24 publications

(16 citation statements)

References 47 publications

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“…X-ray crystallographic study of RedOx active metallo-proteins is challenging as X-ray induced photoreduction can occur. Transition metals are particularly sensitive to specific radiation damage (28, 39), and observation of the FutA Fe(III) state required SFX / neutron diffraction. Changes in the oxidation state induced by X-rays were previously documented for doses as low as 30 - 20 kGy (30, 40, 41).…”

Section: Discussionmentioning

confidence: 99%

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron binding protein FutA fromProchlorococcus

Bolton

Machelett

Stubbs

et al. 2023

Preprint

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The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind ferric (Fe3+), while cytosolic FutA1 binds ferrous (Fe2+). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a solvent molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell was consistent with an overall charge-neutral ferric binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the ferrous binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral ferrous binding site. Room temperature dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between ferric and ferrous states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

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Section: Discussionmentioning

confidence: 99%

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron binding protein FutA fromProchlorococcus

Bolton

Machelett

Stubbs

et al. 2023

Preprint

Self Cite

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“…Large‐scale batch crystallisation to form microcrystals of the desired P450 peroxygenase could be achieved by incorporating ammonium sulfate precipitation to the vapor diffusion crystallisation method [26] . Protein microcrystals are susceptible to damage to X‐ray radiation, [25] but large batches of them can be used for serial crystallography using X‐ray free electron lasers (XFEL) [12d] . As diffraction is essentially instantaneous in serial crystallography this method could obtain diffraction data to explore femtosecond resolved structures along the catalytic pathway of enzymes.…”

Section: Discussionmentioning

confidence: 99%

An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase**

Lee,

Bruning,

Bell

2023

Chemistry A European J

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The cytochrome P450 monooxygenases (CYPs) are a class of heme‐thiolate enzymes that insert oxygen into unactivated C‐H bonds. These enzymes can be converted into peroxygenases via protein engineering which enables their activity to occur using hydrogen peroxide (H2O2) without the requirement for additional nicotinamide co‐factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H2O2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4‐methoxybenzoic acid were soaked with different concentrations of H2O2 for varying times to initiate the in crystallo O‐demethylation reaction. Crystal structures of T252E‐CYP199A4 showed a distinct loss of electron density that was consistent with the O‐demethylated metabolite, 4‐hydroxybenzoic acid. A new X‐ray crystal structure of this enzyme with the 4‐hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate‐bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to intitiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H2O2 will enable new structural biology methods and techniques to be applied to study their mechanism of action.

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“…X-ray Free Electron Lasers (XFELs) produce pulses of coherent X-rays with peak brightness that is nine orders of magnitude greater than synchrotron storage ring radiation and last only 10–100 femtoseconds [ 7 , 65 ]. The field of XFEL macromolecular crystallography has been extensively reviewed [ 38 , 65 , 66 ] and thus we only touch on a few pertinent points here. Because each pulse of XFEL radiation contains ~10 12 photons, diffraction data can be collected serially from a large number of microcrystals, each of which is typically destroyed by these high intensity X-ray pulses [ 67 – 69 ].…”

Section: New X-ray Crystallographic Approaches To Study Cys Chemistrymentioning

confidence: 99%

Understanding Cysteine Chemistry Using Conventional and Serial X-ray Protein Crystallography

Smith

Wilson

2022

Crystals

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Proteins that use cysteine residues for catalysis or regulation are widely distributed and intensively studied, with many biomedically important examples. Enzymes where cysteine is a catalytic nucleophile typically generate covalent catalytic intermediates whose structures are important for understanding mechanism and for designing targeted inhibitors. The formation of catalytic intermediates can change enzyme conformational dynamics, sometimes activating protein motions that are important for catalytic turnover. However, these transiently populated intermediate species have been challenging to structurally characterize using traditional crystallographic approaches. This review describes the use and promise of new time-resolved serial crystallographic methods to study cysteine-dependent enzymes, with a focus on the main (Mpro) and papain-like (PLpro) cysteine proteases of SARS-CoV-2, as well as on other examples. We review features of cysteine chemistry that are relevant for the design and execution of time-resolved serial crystallography experiments. In addition, we discuss emerging X-ray techniques, such as time-resolved sulfur X-ray spectroscopy, that may be able to detect changes in sulfur charge states and covalency during catalysis or regulatory modification. In summary, cysteine-dependent enzymes have features that make them especially attractive targets for new time-resolved serial crystallography approaches, which can reveal both changes to enzyme structures and dynamics during catalysis in crystalline samples.

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Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis

Cited by 24 publications

References 47 publications

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron binding protein FutA fromProchlorococcus

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron binding protein FutA fromProchlorococcus

An In Crystallo Reaction with an Engineered Cytochrome P450 Peroxygenase**

Understanding Cysteine Chemistry Using Conventional and Serial X-ray Protein Crystallography

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