In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2→ S3transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2→ S3transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QAand QB, are observed. At the donor site, tyrosine YZand His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2→ S3transition mirrors the appearance of OXelectron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site (“H-cluster”) consists of a [4Fe–4S]H cluster linked through a bridging cysteine to a [2Fe]H subsite coordinated by CN− and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fed). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe–H species fully consistent with widely accepted models of the catalytic cycle.

Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S2 to S3 transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.

Hydrogenases catalyze the redox interconversion of protons and H 2 , an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO) 2 (CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe 2+ binding and synthon release.FeFe hydrogenase | metallocofactor biosynthesis | HydG F eFe hydrogenases catalyze the reversible interconversion of H 2 with protons and electrons, and thereby provide either an electron source or an electron sink for a variety of metabolic processes (1). Hydrogenase reactivity occurs at the H cluster, which consists of a conventional [4Fe-4S] H subcluster coupled to an organometallic [2Fe] H subcluster that features a 2-aza-1,3-propanedithiolate ("azadithiolate") ligand and multiple CO and CN -ligands (Fig. 1A) (2, 3). The biosynthesis of the H cluster has garnered much attention (4, 5) given its unusual structure and exceptional H 2 production activity (6). Whereas the [4Fe-4S] H subcluster is inserted by the housekeeping Fe-S cluster machinery, the [2Fe] H subcluster is synthesized and inserted by three accessory proteins: the HydE, HydF, and HydG maturases (5, 7-9). Both L-tyrosine (Tyr) and L-cysteine (Cys) have been shown to stimulate in vitro [2Fe] H subcluster biosynthesis (10, 11) with Tyr serving as the precursor to the CO and CN -ligands (12-14); the role of Cys in H-cluster maturation is less clear and an emerging area of focus (15).Significant progress has been made toward elucidating the individual functions of the maturases (5). HydG is a member of the radical S-adenosyl-L-methionine (SAM) family of enzymes (16) (Fig. 1B). The substrate and product of the radical SAM enzyme HydE are presently unknown, although it is thought that HydE plays a role in building the azadithiolate ligand (5, 15). HydG and HydE are thought to function in concert with the GTP-hydrolyzing enzyme HydF (18, 19) to generate a [2Fe] H subcluster-like precursor (20-22) that is transferred to the hydrogenase apoprotein (apo-HydA) to yield the mature H cluster. This mechanistic framework continues to undergo substantial refinement as the chemical details of these processes are unraveled.HydG contains two Fe-S clusters that play separate roles in building the [Fe(CO) 2 (CN)] synthon (17,(23)(24)(25)(26). Cleavage of Tyr to CO and CN -is initiated at the N-terminal, SAM-binding [4Fe-4S] RS cluster where one-electron reduction of SAM generates the 5′-deoxyadenosyl radical (5′-dAdo•) (Fig. 1B). Subsequent H-atom abstraction from the amino group (27) of Tyr (28) induces Cα-Cβ bond cleavage. The resulting 4-hydroxybenzyl radical (4HOB•) has been observed by EP...

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) and Density Functional Theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, with the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of the enzyme in pure oxidation states. Here, microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≤35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage-free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively 57Fe-labeled binuclear subsite is described. The precursor [57Fe2(adt)(CN)2(CO)4]2– was synthesized from the 57Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[57Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary 57Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with 57FeCl3 and Na2S. The Hox-CO state of [257Fe]H and [457Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.

Three maturase enzymes—HydE, HydF, and HydG—synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)]− species is a key intermediate in H-cluster maturation.