Final Stages in the Biosynthesis of the [FeFe]-Hydrogenase Active Site.

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe]H active site of the [FeFe]-hydrogenases. The recently hypothesized intermediate [Fe2(SCH2NH2)2(CN)2(CO)4]2- ([1]2-) was prepared by a multistep route from [Fe2(S2)(CN)(CO)5]-. The following synthetic intermediates were characterized in order: [Fe2(SCH2NHFmoc)2(CNBEt3)(CO)5]-, [Fe2(SCH2NHFmoc)2(CN)-(CO)5]-, and [Fe2(SCH2NHFmoc)2(CN)2(CO)4]2-, where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)]+, PPh4 + and PPN+ salts as well as the 13CD2-isotopologues. These Fe2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial-equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [1]2- maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF.

Hybrids of [FeFe]- and [NiFe]-H2ase Active Site Models.

Complexes of the type (diphosphine)Ni(µ-SR)2Fe(CO)3 are investigated with azadithiolate (adt, HN(CH2S-)2) as the dithiolate. The resulting complexes are hybrid models for the active sites of the [NiFe]- and [FeFe]-hydrogenases. The key complex (dppv)Ni(µ-adt)Fe(CO)3 (3) was prepared from the complex Ni[(SCH2)2NCbz](dppv), which contains a Cbz-protected adt ligand (Cbz = C(O)OCH2Ph, dppv = cis-1,2-(Ph2P)2C2H2). This complex combines with Fe2(CO)9 to give (dppv)Ni[(µ-SCH2)2NCbz]Fe(CO)3, which is readily deprotected to give 3. Complex 3 undergoes protonation at both Fe and N to give successively [(dppv)Ni(µ-adt)FeH(CO)3]+ ([H3]+) and [(dppv)Ni(µ-adtH)FeH(CO)3]2+ ([H3H]2+). The redox properties and dynamics of these complexes resemble previously reported analogues with propanedithiolate. Solutions of [H3]+ readily degrade to [(dppv)Ni[(µ-SCH2)2NCH2]Fe(CO)3]+ ([4]+), which features a methylene group linking N and Fe. Complex [4]+ can be made in high yield by reaction of [H3]+ with CH2O, and this conversion was also demonstrated with 13CH2O. Complex [4]+ undergoes hydrogenolysis by photochemical reaction with H2 to give [(dppv)Ni[(µ-SCH2)2NMe]FeH(CO)3]+, the N-methylated analogue of [H3]+. Upon treatment ith Me3O+, [4]+ undergoes quaternization, giving [(dppv)Ni[(µ-SCH2)2N(Me)CH2]Fe(CO)3]2+. In contrast with the lability of [H3]+, the phosphine-substituted derivative [(dppv)Ni(µ-adt)FeH(CO)2(PPh3)]+ did not degrade. Most complexes were characterized by X-ray crystallography.

Synthesis, Spectroscopy, and Structure of [FeRu(µ-dithiolate)(CN)2(CO)4]2.

The salt [K(18-crown-6)]2[Ru(CN)2(CO)3] ([K(18-crown-6)]2[1]) was generated by the reaction of Ru(C2H4)(CO)4 with [K(18-crown-6)]CN. An initial thermal reaction gives [Ru(CN)(CO)4]-, which, upon ultraviolet (UV) irradiation, reacts with a second equiv of CN-. Protonation of [1]2- gave [HRu(CN)2(CO)3]- ([H1]-), which was isolated as a single isomer with mutually trans cyanide ligands. The complex cis,cis,cis-[Ru(pdt)(CN)2(CO)2]2- ([2]2-) was prepared by the UV-induced reaction of [1]2- with propanedithiol (pdtH2). The corresponding iron complex cis,cis,cis-[Fe(pdt)(CN)2(CO)2]2- ([3]2-) was prepared similarly. The pdt complexes [2]2- and [3]2- were treated with Fe(benzylideneacetone)(CO)3 to give, respectively, [RuFe (µ-pdt)(CN)2(CO)4]2- ([5]2-) and [Fe2(µ-pdt)(CN)2(CO)4]2- ([4]2-). The pathway from [3]2- to Fe2 complex [4]2- implicates intermetallic migration of CN-. In contrast, the formation of [5]2- leaves the Ru(CN)2(CO) center intact, as confirmed by X-ray crystallography. The structure of [5]2- features a "rotated" square-pyramidal Fe(CO)2(µ-CO) site. NMR measurements indicate that the octahedral Ru site is stereochemically rigid, whereas the Fe site dynamically undergoes turnstile rotation. 57Fe Mössbauer spectral parameters are very similar for rotated [5]2- and unrotated Fe2 complex [4]2-, indicating the insensitivity of that technique to both the geometry and the oxidation state of the Fe site. According to cyclic voltammetry, [5]2- oxidizes at E1/2 ∼ -0.8 V vs Fc+/0. Electron paramagnetic resonance (EPR) measurements show that 1e- oxidation of [5]2- gives an S = 1/2 rhombic species, consistent with the formulation Ru(II)Fe(I), related to the Hox state of the [FeFe] hydrogenases. Density functional theory (DFT) studies reproduce the structure, 1H NMR shifts, and infrared (IR) spectra observed for [5]2-. Related homometallic complexes with both cyanides on a single metal are predicted to not adopt rotated structures. These data suggest that [5]2- is best described as Ru(II)Fe(0). This conclusion raises the possibility that for some reduced states of the [FeFe]-hydrogenases, the [2Fe]H site may be better described as Fe(II)Fe(0) than Fe(I)Fe(I).

Characterizing the Biosynthesis of the [Fe(II)(CN)(CO)2(cysteinate)]- Organometallic Product of the Radical-SAM Enzyme HydG by EPR and Mössbauer Spectroscopy.

[FeFe]-hydrogenases employ a catalytic H-cluster, consisting of a [4Fe-4S]H cluster linked to a [2Fe]H subcluster with CO, CN- ligands, and an azadithiolate bridge, which mediates the rapid redox interconversion of H+ and H2. In the biosynthesis of this H-cluster active site, the radical S-adenosyl-l-methionine (radical SAM, RS) enzyme HydG plays the crucial role of generating an organometallic [Fe(II)(CN)(CO)2(cysteinate)]- product that is en route to forming the H-cluster. Here, we report direct observation of this diamagnetic organometallic Fe(II) complex through Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s-1 and quadrupole splitting of ΔEQ = 0.66 mm s-1. These Mössbauer values are a change from the starting values of δ = 1.15 mm s-1 and ΔEQ = 3.23 mm s-1 for the ferrous "dangler" Fe in HydG. These values of the observed product complex B are in good agreement with Mössbauer parameters for the low-spin Fe2+ ions in synthetic analogues, such as 57Fe Syn-B, which we report here. These results highlight the essential role that HydG plays in converting a resting-state high-spin Fe(II) to a low-spin organometallic Fe(II) product that can be transferred to the downstream maturase enzymes.

Fully Refined Semisynthesis of the [FeFe] Hydrogenase H-Cluster.

[FeFe] hydrogenases contain a 6-Fe cofactor that serves as the active site for efficient redox interconversion between H2 and protons. The biosynthesis of the so-called H-cluster involves unusual enzymatic reactions that synthesize organometallic Fe complexes containing azadithiolate, CO, and CN- ligands. We have previously demonstrated that specific synthetic [Fe(CO)x(CN)y] complexes can be used to functionally replace proposed Fe intermediates in the maturation reaction. Here, we report the results from performing such cluster semisynthesis in the context of a recent fully defined cluster maturation procedure, which eliminates unknown components previously employed from Escherichia coli cell lysate and demonstrate this provides a concise route to H-cluster synthesis. We show that formaldehyde can be used as a simple reagent as the carbon source of the bridging adt ligand of H-cluster in lieu of serine/serine hydroxymethyltransferase. In addition to the actual H-cluster, we observe the formation of several H-cluster-like species, the identities of which are probed by cryogenic photolysis combined with EPR/ENDOR spectroscopy.

Synthesis and Dynamics of Ferrous Polychalcogenides [Fe(Ex)(CN)2(CO)2]2- (E = S, Se, or Te).

Elemental chalcogens react with [Fe(CN)2(CO)3]2- to give the following ferrous derivatives: [K(18-crown-6)]2[Fe(S5)(CN)2(CO)2], [K(18-crown-6)]2[Fe(S2)(CN)2(CO)2], [K(18-crown-6)]2[Fe(Se4)(CN)2(CO)2], [K(18-crown-6)]2[Fe(Te2)(CN)2(CO)2], and (NEt4)2[Fe(Te2)(CN)2(CO)2]. While these complex anions crystallized in a single stereochemistry (i.e., trans dicyanides or cis dicyanides), they isomerize in solution upon irradiation. The results are benchmarked by the corresponding studies on benzyl thiolate [K(18-crown-6)]2[Fe(SBn)2(CN)2(CO)2].

Organometallic Fe2(µ-SH)2(CO)4(CN)2 Cluster Allows the Biosynthesis of the [FeFe]-Hydrogenase with Only the HydF Maturase.

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H2. The biosynthesis of the [2Fe]H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO)2(CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe2(µ-SH)2(CN)2(CO)4]2- active HydA1 can be biosynthesized without maturases HydG and HydE.

Biosynthesis of the [FeFe] hydrogenase H-cluster via a synthetic [Fe(II)(CN)(CO)2(cysteinate)]- complex.

The H-cluster of [Fe-Fe] hydrogenase consists of a [4Fe]H subcluster linked by the sulfur of a cysteine residue to an organometallic [2Fe]H subcluster that utilizes terminal CO and CN ligands to each Fe along with a bridging CO and a bridging SCH2NHCH2S azadithiolate (adt) to catalyze proton reduction or hydrogen oxidation. Three Fe-S "maturase" proteins, HydE, HydF, and HydG, are responsible for the biosynthesis of the [2Fe]H subcluster and its incorporation into the hydrogenase enzyme to form this catalytically active H-cluster. We have proposed that HydG is a bifunctional enzyme that uses S-adenosylmethione (SAM) bound to a [4Fe-4S] cluster to lyse tyrosine via a transient 5'-deoxyadenosyl radical to produce CO and CN ligands to a unique cysteine-chelated Fe(II) that is linked to a second [4Fe-4S] cluster via the cysteine sulfur. In this "synthon model", after two cycles of tyrosine lysis, the product of HydG is completed: a [Fe(CN)(CO)2(cysteinate)]- organometallic unit that is vectored directly into the synthesis of the [2Fe]H sub-cluster. However our HydG-centric synthon model is not universally accepted, so further validation is important. In this Frontiers article, we discuss recent results using a synthetic "Syn-B" complex that donates [Fe(CN)(CO)2(cysteinate)]- units that match our proposed HydG product. Can Syn-B activate hydrogenase in the absence of HydG and its tyrosine substrate? If so, since Syn-B can be synthesized with specific magnetic nuclear isotopes and with chemical substitutions, its use could allow its enzymatic conversions on the route to the H-cluster to be monitored and modeled in fresh detail.

Surprising Condensation Reactions of the Azadithiolate Cofactor.

Azadithiolate, a cofactor found in all [FeFe]-hydrogenases, is shown to undergo acid-catalyzed rearrangement. Fe2 [(SCH2 )2 NH](CO)6 self-condenses to give Fe6 [(SCH2 )3 N]2 (CO)17 . The reaction, which is driven by loss of NH4+ , illustrates the exchange of the amine group. X-ray crystallography reveals that three Fe2 (SR)2 (CO)x butterfly subunits interconnected by the aminotrithiolate [N(CH2 S)3 ]3- . Mechanistic studies reveal that Fe2 [(SCH2 )2 NR](CO)6 participate in a range of amine exchange reactions, enabling new methodologies for modifying the adt cofactor. Ru2 [(SCH2 )2 NH](CO)6 also rearranges, but proceeds further to give derivatives with Ru-alkyl bonds Ru6 [(SCH2 )3 N][(SCH2 )2 NCH2 ]S(CO)17 and [Ru2 [(SCH2 )2 NCH2 ](CO)5 ]2 S.

Homoleptic Rhodium Pyridine Complexes for Catalytic Hydrogen Oxidation.

The homoleptic rhodium pyridine complex [Rh(py)4]+ ([1]+) is prepared from simple precursors. Lacking good π-acceptor ligands but being sterically protected, [1]+ reversibly oxidizes to colorless [Rh(py)4(thf)2]2+. This monomeric S = 1/2 Rh(II) complex activates H2 to give [HRh(py)4L]2+, which can also be generated by protonation of [1]+. The Rh(III)-H bond is weak, being susceptible to H atom abstraction as well as deprotonation. These results underpin a novel catalytic system for the oxidation of H2 by ferrocenium.

Crystal Structure of the [FeFe]-Hydrogenase Maturase HydE Bound to Complex-B.

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN- ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (-S-CH2-NH-CH2-S-) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN- to produce a unique l-cysteine-Fe(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric "SFe(CO)2CN" precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by 13C2H-ADT Labeling.

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe]H subcluster. A -13C2H2- ADT labeling in the synthetic diiron precursor of [2Fe]H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), and NRVS was measured on samples poised in the catalytically crucial Hhyd state containing a terminal hydride at the distal Fe site. The 13C2H isotope effects were observed also in the Hhyd spectrum. DFT simulations of the spectra allowed identification of the 57Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H2 formation.

CO substitution by PPh3 in Fe2S2(CO)6 proceeds via a novel Fe2S intermediate.

The reaction of Fe2S2(CO)6 and PPh3 affords Fe2S2(CO)4(PPh3)2 by an unprecedented mechanism involving the intermediacy of SPPh3 and Fe2S(CO)6(PPh3)2.

Computational and Experimental Investigations of the Fe2(µ-S2)/Fe2(µ-S)2 Equilibrium.

Density functional theory (DFT) calculations on Fe2S2(CO)6-2n(PMe3)2n for n = 0, 1, and 2 reveal that the most electron-rich derivatives (n = 2) exist as diferrous disulfides lacking an S-S bond. The thermal interconversion of the FeII2(S)2 and FeI2(S2) valence isomers is symmetry-forbidden. Related electron-rich diiron complexes [Fe2S2(CN)2(CO)4]2- of an uncertain structure are implicated in the biosynthesis of [FeFe]-hydrogenases. Several efforts to synthesize electron-rich derivatives of Fe2(µ-S2)(CO)6 (1) are described. First, salts of iron persulfido cyanides [Fe2(µ-S2)(CO)5(CN)]- and [Fe2(µ-S2)(CN)(CO)4(PPh3)]- were prepared by the reactions of NaN(tms)2 with 1 and Fe2(µ-S2)(CO)5(PPh3), respectively. Alternative approaches to electron-rich diiron disulfides targeted Fe2(µ-S2)(CO)4(diphosphine). Whereas the preparation of Fe2(µ-S2)(CO)4(dppbz) was straightforward, that of Fe2(µ-S2)(CO)4(dppv) required an indirect route involving the oxidation of Fe2(µ-SH)2(CO)4(dppv) (dppbz = C6H4-1,2-(PPh2)2, dppv = cis-C2H2(PPh2)2). DFT calculations indicate that the oxidation of Fe2(µ-SH)2(CO)4(dppv) produces singlet diferrous disulfide Fe2(µ-S)2(CO)4(dppv), which is sufficiently long-lived as to be trapped by ethylene. The reaction of 1 and dppv mainly afforded Fe2(µ-SCH=CHPPh2)(µ-SPPh2)(CO)5, implicating a S-centered reaction.

Challenges in the Synthesis of Active Site Mimics for [NiFe]-Hydrogenases.

One of the more active areas in bioorganometallic chemistry is the preparation and reactivity studies of active site mimics of the [NiFe]-hydrogenases. One area of particular recent progress involves reactions that interconvert Ni(µ-X)Fe centers for X = OH, H, CO, as described by Song et al. Such reactions illustrate new ways to access intermediates related to the Ni-R and Ni-SI states of the enzyme. Most models are derivatives of the type (diphosphine)Ni(SR)2Fe(CO)3-n(PR'3)n. In recent work, the methodology has been generalized to include FeII(diphosphine) derivatives of Ni(N2S2), where N2S22- is the tetradentate diamine-dithiolate (CH2N(CH3)CH2CH2S-)2. Indeed, models based on Ni(N2S2) have proven valuable, but these studies also highlight challenges in working with heterobimetallic complexes, specifically the tendency of some such Ni-Fe complexes to convert to homometalliic Ni-Ni derivatives. This kind of problem is not readily detected by X-ray crystallography. With this caution in mind, we argue that one series of complexes recently described in this journal are almost certainly misassigned.

Inhibition of [FeFe]-hydrogenase by formaldehyde: proposed mechanism and reactivity of FeFe alkyl complexes.

The mechanism for inhibition of [FeFe]-hydrogenases by formaldehyde is examined with model complexes. Key findings: (i) CH2 donated by formaldehyde covalently link Fe and the amine cofactor, blocking the active site and (ii) the resulting Fe-alkyl is a versatile electrophilic alkylating agent. Solutions of Fe2[(µ-SCH2)2NH](CO)4(PMe3)2 (1) react with a mixture of HBF4 and CH2O to give three isomers of [Fe2[(µ-SCH2)2NCH2](CO)4(PMe3)2]+ ([2]+). X-ray crystallography verified the NCH2Fe linkage to an octahedral Fe(ii) site. Although [2]+ is stereochemically rigid on the NMR timescale, spin-saturation transfer experiments implicate reversible dissociation of the Fe-CH2 bond, allowing interchange of all three diastereoisomers. Using 13CH2O, the methylenation begins with formation of [Fe2[(µ-SCH2)2N13CH2OH](CO)4(PMe3)2]+. Protonation converts this hydroxymethyl derivative to [2]+, concomitant with 13C-labelling of all three methylene groups. The Fe-CH2N bond in [2]+ is electrophilic: PPh3, hydroxide, and hydride give, respectively, the phosphonium [Fe2[(µ-SCH2)2NCH2PPh3](CO)4(PMe3)2]+, 1, and the methylamine Fe2[(µ-SCH2)2NCH3](CO)4(PMe3)2. The reaction of [Fe2[(µ-SCH2)2NH](CN)2(CO)4]2- with CH2O/HBF4 gave [Fe2[(µ-SCH2)2NCH2CN](CN)(CO)5]- ([4]-), the result of reductive elimination from [Fe2[(µ-SCH2)2NCH2](CN)2(CO)4]-. The phosphine derivative [Fe2[(µ-SCH2)2NCH2CN](CN)(CO)4(PPh3)]- ([5]-) was characterized crystallographically.

Using nature's blueprint to expand catalysis with Earth-abundant metals.

Numerous redox transformations that are essential to life are catalyzed by metalloenzymes that feature Earth-abundant metals. In contrast, platinum-group metals have been the cornerstone of many industrial catalytic reactions for decades, providing high activity, thermal stability, and tolerance to chemical poisons. We assert that nature's blueprint provides the fundamental principles for vastly expanding the use of abundant metals in catalysis. We highlight the key physical properties of abundant metals that distinguish them from precious metals, and we look to nature to understand how the inherent attributes of abundant metals can be embraced to produce highly efficient catalysts for reactions crucial to the sustainable production and transformation of fuels and chemicals.

Radical SAM Enzyme HydE Generates Adenosylated Fe(I) Intermediates En Route to the [FeFe]-Hydrogenase Catalytic H-Cluster.

The H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S]H-subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe]H-subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe]H-subcluster is assembled by a set of Fe-S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [FeII(Cys)(CO)2(CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5'-deoxyadenosyl radical attacking the cysteine S to form a C5'-S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3-S bond, producing a mononuclear [FeI(CO)2(CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe]H-subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.

Reactions of [Fe6C(CO)14(S)]2-: Cluster Growth, Redox, Sulfiding.

Redox reactions, substitutions, and metalations are reported for the iron carbido sulfide [Fe6C(CO)14(S)]2- ([1]2-). Dianion [1]2- oxidized to [Fe6C(CO)16(S)]0 ([2]0) upon treatment with of [Fe(C5H5)2]BF4 or HBF4 (H2 formation) under an atmosphere of CO. Reaction of [2]0 with tBuNC gave [Fe6C(S)(CO)13(tBuNC)5], consisting of Fe5C(CO)13 and [Fe(tBuNC)5]2+ subunits linked by a µ3-S2-. The Fe7CS cluster [Fe7C(CO)17(S)]2- formed upon treatment of (Ph4P)2[1] with Fe(benzylideneacetone)(CO)3. The Fe7 species is an edge-fused cluster with [Fe6C(CO)10(µ-CO)4] and Fe(CO)3 subunits joined by µ3-S and two Fe-Fe bonds. The analogous reaction using Mo(CO)4(norbornadiene) gave [MoFe6C(CO)18(S)]2-. In this cluster, the Mo center is located in the octahedral subunit. Treatment of [1]2- with SO2 afforded [Fe6C(S)(SO2)(CO)13]2-. This cluster features an Fe6C core decorated with µ3-S and µ2-SO2 ligands. These experiments were undertaken in an effort to connect organometallic clusters to FeMoco.

Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.