Search Results

You searched for all publications:

with publication states 'Published (Approved)'

Search Again...

These results only include publications that have been reviewed and processed by Diamond Light Source. To also view papers that have not yet been processed click here.

You can use the folder icon under Actions to collate papers as required. You can then click on View Folder in the left-hand navigation to review and/or download your folder.

Exact matches
Related results

6 items found (0 items not approved), displaying all items.1

Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I24-Microfocus Macromolecular Crystallography	Mechanistic exploitation of a self-repairing, blocked proton transfer pathway in an O 2 -tolerant [NiFe]-hydrogenase Rhiannon M. Evans , Philip A. Ash , Stephen E. Beaton , Emily J. Brooke , Kylie A. Vincent , Stephen B. Carr , Fraser A. Armstrong Journal Of The American Chemical Society DOI: 10.1021/jacs.8b04798 Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe–3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states “Nia-R” and “Nia-C”, even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, “Ni-B”, is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.	Aug 2018
I03-Macromolecular Crystallography	The Structure of Hydrogenase-2 from Escherichia coli: Implications for H 2 -Driven Proton Pumping Stephen E. Beaton , Rhiannon M. Evans , Alexander J. Finney , Ciaran M Lamont , Fraser A. Armstrong , Frank Sargent , Stephen B. Carr Biochemical Journal DOI: 10.1042/BCJ20180053 Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: Under anaerobic conditions Escherichia coli is able to metabolize molecular hydrogen via the action of several [NiFe]-hydrogenase enzymes. Hydrogenase-2, which is typically present in cells at low levels during anaerobic respiration, is a periplasmic-facing membrane-bound complex that functions as a proton pump to convert energy from H2 oxidation into a proton gradient; consequently, its structure is of great interest. Empirically, the complex consists of a tightly-bound core catalytic module, comprising large (HybC) and small (HybO) subunits, which is attached to an Fe-S protein (HybA) and an integral membrane protein, HybB. To date, efforts to gain a more detailed picture have been thwarted by low native expression levels of hydrogenase-2 and the labile interaction between HybOC and HybA/HybB subunits. In this paper we describe a new over-expression system that has facilitated determination of high-resolution crystal structures of HybOC and, hence, a prediction of the quaternary structure of the HybOCAB complex.	Mar 2018
B22-Multimode InfraRed imaging And Microspectroscopy	Generating single metalloprotein crystals in well-defined redox states: electrochemical control combined with infrared imaging of a NiFe hydrogenase crystal Philip A. Ash , Stephen B. Carr , Holly A. Reeve , Aiste Skorupskaite , Jack Rowbotham , Rebecca Shutt , Mark Frogley , Rhiannon Mari Evans , Gianfelice Cinque , Fraser A Armstrong , Kylie A Vincent Chem. Commun. DOI: 10.1039/C7CC02591B Diamond Proposal Number(s): [13879] Show Abstract ▼ Abstract: We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchtroton infrared microspectroscopic imaging. For NiFe hydrogenase 1 from Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.	May 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	Importance of the Active Site “Canopy” Residues in an O 2 -Tolerant [NiFe]-Hydrogenase Emily J. Brooke , Rhiannon M. Evans , Shams T. A. Afroza Islam , Gerri M. Roberts , Sara A. M. Wehlin , Stephen B. Carr , Simon E. V. Phillips , Fraser A. Armstrong Biochemistry DOI: 10.1021/acs.biochem.6b00868 Diamond Proposal Number(s): [12346] Show Abstract ▼ Abstract: The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a “canopy” above the bimetallic center, closest to the site at which exogenous agents CO and O2 interact, substrate H2 binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H2 activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N–metal distance of 4.4 Å): its replacement with lysine lowers the H2 oxidation rate by nearly 2 orders of magnitude and markedly decreases the H2/D2 kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H2 oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H2 oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O2 tolerance by stabilizing the oxidized resting NiIII–OH state (“Ni-B”). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.	Dec 2016
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Hydrogen activation by [NiFe]-hydrogenases S. B. Carr , R. M. Evans , E. J. Brooke , S. A. M. Wehlin , E. Nomerotskaia , F. Sargent , F. A. Armstrong , S. E. V. Phillips Biochemical Society Transactions 44, 863 - 868 DOI: 10.1042/BST20160031 Diamond Proposal Number(s): [9306] Show Abstract ▼ Abstract: Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H-2. The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg(509)), which interacts with two conserved aspartate residues (Asp(118) and Asp(574)) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu(28)) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H-2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H-2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg(509) (the base).	Jun 2016
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Mechanism of hydrogen activation by [NiFe] hydrogenases Rhiannon M Evans , Emily J Brooke , Sara A M Wehlin , Elena Nomerotskaia , Frank Sargent , Stephen B Carr , Simon E V Phillips , Fraser A Armstrong Nature Chemical Biology DOI: 10.1038/nchembio.1976 PMID: 26619250 Diamond Proposal Number(s): [9306] Show Abstract ▼ Abstract: The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom <4.5 Å above the nickel and iron atoms. The guanidine headgroup interacts with the side chains of two conserved aspartic acid residues to complete an outer-shell canopy that has thus far proved intractable to investigation by site-directed mutagenesis. Using hydrogenase-1 from Escherichia coli, the strictly conserved residues R509 and D574 have been replaced by lysine (R509K) and asparagine (D574N) and the highly conserved D118 has been replaced by alanine (D118A) or asparagine (D118N/D574N). Each enzyme variant is stable, and their [(RS)2Niμ(SR)2Fe(CO)(CN)2] inner coordination shells are virtually unchanged. The R509K variant had >100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H2 activation by [NiFe] hydrogenases.	Nov 2015

Publications Database

Search Results

You searched for all publications:

Search Again...

Subject Areas (check all that apply) select all

Instruments used to collect data (check all that apply) select all

Collaborations involved (check all that apply) select all

Diamond Offline Facilities used

Relevant Technical Areas (check all that apply) select all

Menu for Anonymous User