Simon J. George

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydFEG), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydFEG. The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.

(FeFe)-Hydrogenase Maturation: HydG-Catalyzed Synthesis of Carbon Monoxide

Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.

High-yield expression of heterologous [FeFe] hydrogenases in Escherichia coli.

Background The realization of hydrogenase-based technologies for renewable H2 production is presently limited by the need for scalable and high-yielding methods to supply active hydrogenases and their required maturases. Principal Findings In this report, we describe an improved Escherichia coli-based expression system capable of producing 8–30 mg of purified, active [FeFe] hydrogenase per liter of culture, volumetric yields at least 10-fold greater than previously reported. Specifically, we overcame two problems associated with other in vivo production methods: low protein yields and ineffective hydrogenase maturation. The addition of glucose to the growth medium enhances anaerobic metabolism and growth during hydrogenase expression, which substantially increases total yields. Also, we combine iron and cysteine supplementation with the use of an E. coli strain upregulated for iron-sulfur cluster protein accumulation. These measures dramatically improve in vivo hydrogenase activation. Two hydrogenases, HydA1 from Chlamydomonas reinhardtii and HydA (CpI) from Clostridium pasteurianum, were produced with this improved system and subsequently purified. Biophysical characterization and FTIR spectroscopic analysis of these enzymes indicate that they harbor the H-cluster and catalyze H2 evolution with rates comparable to those of enzymes isolated from their respective native organisms. Significance The production system we describe will facilitate basic hydrogenase investigations as well as the development of new technologies that utilize these prolific H2-producing enzymes. These methods can also be extended for producing and studying a variety of oxygen-sensitive iron-sulfur proteins as well as other proteins requiring anoxic environments.

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

Three iron-sulfur proteins–HydE, HydF, and HydG–play a key role in the synthesis of the [2Fe]H component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-l-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN− ligands of the [2Fe]H cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN− ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the 13C, 15N, and 57Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe]H component of the H-cluster. Vibrational spectroscopy traces the origin of carbon monoxide and cyanide ligands in the active site of di-iron hydrogenase enzymes. [Also see Perspective by Pickett] Sourcing CO and Cyanide Hydrogenase enzymes derive their activity in part from the coordination of CO and cyanide ligands to metals in their active sites. Recent work elucidated the jettisoning of a tyrosine side chain at the outset of the biosynthetic pathway toward these ligands in the di-iron class of hydrogenase. Kuchenreuther et al. (p. 424; see the Perspective by Pickett) now apply stopped-flow infrared spectroscopy to uncover the next portion of the pathway, during which the residual tyrosine fragment is further broken down into CO and CN− ligands at a single iron center in an iron sulfur cluster associated with the HydG enzyme.

A Radical Intermediate in Tyrosine Scission to the CO and CN− Ligands of FeFe Hydrogenase

Piecing Together Hydrogenase Microbial hydrogenase enzymes generally use iron to catalyze the reversible formation of hydrogen from protons and electrons. Key to their efficiency is a set of iron-coordinating ligands, including CO and cyanide. Kuchenreuther et al. (p. 472) examined how the HydG maturase enzyme breaks down the amino acid tyrosine to derive these diatomic ligands for assembly of the diiron class of hydrogenases. The first step involves abstraction of an H atom from the phenolic OH substituent of the side chain. Electron paramagnetic resonance spectroscopy revealed a radical intermediate that subsequently results from heterolysis of the bond tethering the side chain to the α-carbon. With the side chain thus jettisoned, the residual dehydroglycine could be transformed into CO and CN−. Electron paramagnetic resonance spectroscopy elucidates a key step in the biosynthesis of hydrogenase active site ligands. The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN− for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of 2H, 13C, and 15N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cβ bond forms a transient 4-oxidobenzyl (4OB•) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB• radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN−, a key intermediate in the assembly of the 2Fe subunit of the H cluster.

Cell-free H-cluster Synthesis and [FeFe] Hydrogenase Activation: All Five CO and CN− Ligands Derive from Tyrosine

[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN−) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN− ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

Variable-temperature magnetic circular dichroism

Publisher Summary A magnetic circular dichroism (MCD) spectrum is a measurement of the difference between the absorption of left and right circularly polarized light as a function of the wavelength of the measuring beam when a sample is placed in a magnetic field applied parallel or antiparallel to the direction of propagation of the light beam. There are three possible consequences to the application of a magnetic field—namely, Zeeman splitting of ground and/or excited degenerate states, field-induced mixing of states, and a change in the population of molecules over the Zeeman sublevels of a paramagnetic ground state. These give rise to contributions to the intensity of an MCD spectrum called, respectively, the A-term, the B-term, and the C-term. This chapter describes in detail for the first time the methodology used in the Norwich laboratory (University of East Anglia), which has become that almost universally employed, for the measurement of MCD spectra in the low-temperature and high-field regions. The analysis of magnetization curves is discussed and examples given briefly of the spectra of some of the main classes of transition metal centers.

Direct electrochemistry in the characterisation of redox proteins: Novel properties of Azotobacter 7Fe ferredoxin

Fast diffusion‐dominated electron transfer between Azotobacter chroococcum 7Fe ferredoxin, FdI, and pyrolytic graphite ‘edge’ electrodes, promoted by aminoglycosides, permits detailed voltammetric studies and preparation of oxidation states inaccessible by chemical titration. The [3Fe‐4S] cluster exhibits pH dependent E values (30°C); E (alkaline) = 460 ± 10 mV vs NHE, −dE /d(pH) = 55 mV, pK = 7.8. The [4Fe‐4S] cluster is characterised by an unusually low reduction potential, −645 ± 10 mV vs NHE, at pH 8.3, with a slight pH dependence, −dE /d(pH) ∼25 mV over the pH range 8.5‐7.0. No redox couple is observed at potentials between −300 and +600 mV vs NHE. This shows that the [4Fe‐4S] cluster is not an HIPIP‐type centre. The electron paramagnetic resonance spectrum, centred at g = 1.93, of the product resulting from bulk electrolysis at −835 mV is assigned to a [4Fe‐4S]+ cluster interacting magnetically with a reduced [3Fe‐4S] cluster.

Extended X-ray Absorption Fine Structure and Nuclear Resonance Vibrational Spectroscopy Reveal that NifB-co, a FeMo-co Precursor, Comprises a 6Fe Core with an Interstitial Light Atom

NifB-co, an Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway to the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy together with (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. The spectra have been interpreted in part by comparison with data for the completed FeMo-co attached to the NafY carrier protein: the NafY:FeMo-co complex. EXAFS analysis of the NifX:NifB-co complex yields an average Fe-S distance of 2.26 A and average Fe-Fe distances of 2.66 and 3.74 A. Search profile analyses reveal the presence of a single Fe-X (X = C, N, or O) interaction at 2.04 A, compared to a 2.00 A Fe-X interaction found in the NafY:FeMo-co EXAFS. This suggests that the interstitial light atom (X) proposed to be present in FeMo-co has already inserted at the NifB-co stage of biosynthesis. The NRVS exhibits strong bands from Fe-S stretching modes peaking around 270, 315, 385, and 408 cm(-1). Additional intensity at approximately 185-200 cm(-1) is interpreted as a set of cluster breathing modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data for NifX:NifB-co are best simulated using a Fe 6S 9X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. The results are consistent with the conclusion that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.

Nitrogen chemical structure in DNA and related molecules by X-ray absorption spectroscopy

The electronic environment of nitrogen in nucleic acid bases, nucleotides, polynucleotides and DNA has been studied, for the first time using X-Ray Absorption Near-Edge Spectroscopy (XANES). Generally, the spectra of these complex molecules consist of low energy bands corresponding to 1s-->pi* transitions and high energy bands corresponding to 1s-->sigma* transition, as illustrated using several nitrogen model compounds. The 1s-->pi* transitions show particular sensitivity to the chemical environment of the nitrogen. Oxygen substitution on ring carbon atoms generally results in a significant blue shift of the lowest 1s-->pi* bands while halogen substitution results in a small blue shift. These observations illustrate the significance of the disturbance of the aromatic ring system produced by exocyclic carbonyl groups. Direct substitution on the nitrogen frequently results in significant spectral perturbations. Differences between the spectra of the polynucleotides and the sums of spectra of the individual nucleotides point to the effects of hydrogen-bonding in complementary double-helix structures. The XANES spectrum of a DNA sample with a known ratio of the polynucleotides is equivalent to the weighted sum of the spectra of individual polynucleotides, indicating that the difference in base stacking interactions produces negligible spectral effects. The variability of nitrogen K-edge spectra in these samples and in protein may be useful for chemically specific imaging using X-ray microscopes.