The final steps of [FeFe]-hydrogenase maturation.

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.

[FeFe]-Hydrogenases: maturation and reactivity of enzymatic systems and overview of biomimetic models.

While hydrogen plays an ever-increasing role in modern society, nature has utilized hydrogen since a very long time as an energy carrier and storage molecule. Among the enzymatic systems that metabolise hydrogen, [FeFe]-hydrogenases are one of the most powerful systems to perform this conversion. In this light, we will herein present an overview on developments in [FeFe]-hydrogenase research with a strong focus on synthetic mimics and their application within the native enzymatic environment. This review spans from the biological assembly of the natural enzyme and the highly controversial discussed mechanism for the hydrogen generation to the synthesis of multiple mimic platforms as well as their electrochemical behaviour.

How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer.

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases.

Differential Protonation at the Catalytic Six-Iron Cofactor of [FeFe]-Hydrogenases Revealed by 57Fe Nuclear Resonance X-ray Scattering and Quantum Mechanics/Molecular Mechanics Analyses.

[FeFe]-hydrogenases are efficient biological hydrogen conversion catalysts and blueprints for technological fuel production. The relations between substrate interactions and electron/proton transfer events at their unique six-iron cofactor (H-cluster) need to be elucidated. The H-cluster comprises a four-iron cluster, [4Fe4S], linked to a diiron complex, [FeFe]. We combined 57Fe-specific X-ray nuclear resonance scattering experiments (NFS, nuclear forward scattering; NRVS, nuclear resonance vibrational spectroscopy) with quantum-mechanics/molecular-mechanics computations to study the [FeFe]-hydrogenase HYDA1 from a green alga. Selective 57Fe labeling at only [4Fe4S] or [FeFe], or at both subcomplexes was achieved by protein expression with a 57Fe salt and in vitro maturation with a synthetic diiron site precursor containing 57Fe. H-cluster states were populated under infrared spectroscopy control. NRVS spectral analyses facilitated assignment of the vibrational modes of the cofactor species. This approach revealed the H-cluster structure of the oxidized state (Hox) with a bridging carbon monoxide at [FeFe] and ligand rearrangement in the CO-inhibited state (Hox-CO). Protonation at a cysteine ligand of [4Fe4S] in the oxidized state occurring at low pH (HoxH) was indicated, in contrast to bridging hydride binding at [FeFe] in a one-electron reduced state (Hred). These findings are direct evidence for differential protonation either at the four-iron or diiron subcomplex of the H-cluster. NFS time-traces provided Mössbauer parameters such as the quadrupole splitting energy, which differ among cofactor states, thereby supporting selective protonation at either subcomplex. In combination with data for reduced states showing similar [4Fe4S] protonation as HoxH without (Hred') or with (Hhyd) a terminal hydride at [FeFe], our results imply that coordination geometry dynamics at the diiron site and proton-coupled electron transfer to either the four-iron or the diiron subcomplex discriminate catalytic and regulatory functions of [FeFe]-hydrogenases. We support a reaction cycle avoiding diiron site geometry changes during rapid H2 turnover.

Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics.

The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations.

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN-) ligands. Hydrogen (H2) and oxygen (O2) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. 57Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fed in a [4Fe4S-2Fe]3- complex with the net spin on Fed whereas Hox-CO shows an apical CN- at Fed in a [4Fe4S-2Fe(CO)]3- complex with net spin sharing among Fep and Fed (proximal or distal iron ions in [2Fe]). At ambient O2 pressure, a novel H-cluster species (Hox-O2) accumulated in C169A, assigned to a [4Fe4S-2Fe(O2)]3- complex with an apical superoxide (O2-) carrying the net spin bound at Fed. H2 exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]3- complex with the net spin on the reduced cubane, an apical hydride at Fed, and a proton at a cysteine ligand. Hox-O2 and Hhyd are stabilized by impaired O2- protonation or proton release after H2 cleavage due to interruption of the proton path towards and out of the active site.

Protonation/reduction dynamics at the [4Fe-4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases.

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN-) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN- vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN- infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide.

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe⁻4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe⁻4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe⁻Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe⁻4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe⁻4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed.

Bridging Hydride at Reduced H-Cluster Species in [FeFe]-Hydrogenases Revealed by Infrared Spectroscopy, Isotope Editing, and Quantum Chemistry.

[FeFe]-Hydrogenases contain a H2-converting cofactor (H-cluster) in which a canonical [4Fe-4S] cluster is linked to a unique diiron site with three carbon monoxide (CO) and two cyanide (CN-) ligands (e.g., in the oxidized state, Hox). There has been much debate whether reduction and hydrogen binding may result in alternative rotamer structures of the diiron site in a single (Hred) or double (Hsred) reduced H-cluster species. We employed infrared spectro-electrochemistry and site-selective isotope editing to monitor the CO/CN- stretching vibrations in [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. Density functional theory calculations yielded vibrational modes of the diatomic ligands for conceivable H-cluster structures. Correlation analysis of experimental and computational IR spectra has facilitated an assignment of Hred and Hsred to structures with a bridging hydride at the diiron site. Pronounced ligand rotation during µH binding seems to exclude Hred and Hsred as catalytic intermediates. Only states with a conservative H-cluster geometry featuring a µCO ligand are likely involved in rapid H2 turnover.

Interplay between CN- Ligands and the Secondary Coordination Sphere of the H-Cluster in [FeFe]-Hydrogenases.

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4FeH) linked to a binuclear iron-sulfur cluster (2FeH) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2FeH) subsite features a diatomic coordination sphere composed of three CO and two CN- ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN- signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN- ligands tightly anchoring the (2FeH) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN- ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN- ligands of the (2FeH) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN- ligand (i.e., closest to the (4FeH) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

Organometallic Fe-Fe Interactions: Beyond Common Metal-Metal Bonds and Inverse Mixed-Valent Charge Transfer.

The compounds [Fe(CO)3 (dRpf)]n+ , n=0, 1, 2 and dRpf=1,1'-bis(dicyclohexylphosphino)ferrocene ([1]n+ ) or 1,1'-bis(diisopropylphosphino)ferrocene ([2]n+ ), were obtained as two-step reversible redox systems by photolytic and redox reactions. The iron-iron distance decreases from about 4 Što about 3 Šon oxidation, which takes place primarily at the tricarbonyliron moiety. Whereas ferrocene oxidation is calculated to occur only in excited states, the near infrared absorptions of the mixed-valent monocations are due to an unprecedented "inverse" inter-valence charge transfer from the electron-rich iron(II) in the ferrocene backbone to the electron-deficient tricarbonyliron(I). Protonation of complex 1 results in the formation of the structurally characterized hydride [1H]BF4 , which reacts with acetone to form the dication, 12+ , and isopropanol. While the hydride [2H]BF4 was found to be unstable, protonation of 2 in acetone resulted in the clean formation of 22+, formally a hydrogen transfer.

Redox Induced Configurational Isomerization of Bisphosphine-Tricarbonyliron(I) Complexes and the Difference a Ferrocene Makes.

The tricarbonyliron (TCFe) complexes Fe(CO)3(dppf) and Fe(CO)3(dppp), where dppf = 1,1'-bis(diphenylphosphino)ferrocene and dppp = 1,3-bis(diphenylphosphino)propane, exhibit redox activity that induces configurational isomerization. The presence of the ferrocenyl (Fc) group stabilizes higher oxidized forms of TCFe. Using spectroelectrochemistry (IR, UV-vis, Mössbauer, and EPR) and computational analysis, we can show that the Fc in the backbone of the dppf ligand tends to form a weak dative bond to the electrophilic TCFeI and TCFeII species. The open shell TCFeI intermediate was stabilized by the distribution of spin between the two Fe centers (Fc and TCFe), whereas lacking the Fc moiety resulted in highly reactive TCFeI species. The [Fe(CO)3(dppf)]+ cation adopts two possible configurations, square-pyramidal (without an Fe-Fe interaction) and trigonal-bipyramidal (containing an Fe-Fe interaction). The two configurations are in equilibrium with the trigonal-bipyramidal configuration being enthalpically favored (ΔH = -7 kJ mol-1). There is an entropic penalty (ΔS = -20 J mol-1) due to tilting of the Cp (cyclopentadienide) rings of the dppf moieties by ∼8°. Additionally, the terminal iron hydride [FeH(CO)3(dppf)]BF4 was formed by protonation with a strong acid (HBF4·Et2O).

Proton-Coupled Reduction of the Catalytic [4Fe-4S] Cluster in [FeFe]-Hydrogenases.

In nature, [FeFe]-hydrogenases catalyze the uptake and release of molecular hydrogen (H2 ) at a unique iron-sulfur cofactor. The absence of an electrochemical overpotential in the H2 release reaction makes [FeFe]-hydrogenases a prime example of efficient biocatalysis. However, the molecular details of hydrogen turnover are not yet fully understood. Herein, we characterize the initial one-electron reduction of [FeFe]-hydrogenases by infrared spectroscopy and electrochemistry and present evidence for proton-coupled electron transport during the formation of the reduced state Hred'. Charge compensation stabilizes the excess electron at the [4Fe-4S] cluster and maintains a conservative configuration of the diiron site. The role of Hred' in hydrogen turnover and possible implications on the catalytic mechanism are discussed. We propose that regulation of the electronic properties in the periphery of metal cofactors is key to orchestrating multielectron processes.

Electrochemical Investigations of the Mechanism of Assembly of the Active-Site H-Cluster of [FeFe]-Hydrogenases.

Protein film electrochemistry (PFE) has been used to study the assembly of the complex 6Fe active site of [FeFe]-hydrogenases (known as the H-cluster) from its precursors-the [4Fe-4S] domain that is already coordinated within the host, and the 2Fe domain that is presented as a synthetic water-soluble complex stabilized by an additional CO. Not only does PFE allow control of redox states via the electrode potential but also the immobilized state of the enzyme facilitates control of extremely low concentrations of the 2Fe complex. Results for two enzymes, CrHydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum, are very similar, despite large differences in size and structure. Assembly begins with very tight binding of the 34-valence electron 2Fe complex to the apo-[4Fe-4S] enzyme, well before the rate-determining step. The precursor is trapped under highly reducing conditions (<-0.5 V vs SHE) that prevent fusion of the [4Fe-4S] and 2Fe domains (via cysteine-S) since the immediate product would be too electron-rich. Relaxing this condition allows conversion to the active H-cluster. The intramolecular steps are relevant to the final stage of biological H-cluster maturation.

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase.

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H2 oxidation activity, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.

Insights from 125Te and 57Fe nuclear resonance vibrational spectroscopy: a [4Fe-4Te] cluster from two points of view.

Iron-sulfur clusters are common building blocks for electron transport and active sites of metalloproteins. Their comprehensive investigation is crucial for understanding these enzymes, which play important roles in modern biomimetic catalysis and biotechnology applications. We address this issue by utilizing (Et4N)3[Fe4Te4(SPh)4], a tellurium modified version of a conventional reduced [4Fe-4S]+ cluster, and performed both 57Fe- and 125Te-NRVS to reveal its characteristic vibrational features. Our analysis exposed major differences in the resulting 57Fe spectrum profile as compared to that of the respective [4Fe-4S] cluster, and between the 57Fe and 125Te profiles. DFT calculations are applied to rationalize structural, electronic, vibrational, and redox-dependent properties of the [4Fe-4Te]+ core. We herein highlight the potential of sulfur/tellurium exchange as a method to isolate the iron-only motion in enzymatic systems.

Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases.

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

Electronic and molecular structure relations in diiron compounds mimicking the [FeFe]-hydrogenase active site studied by X-ray spectroscopy and quantum chemistry.

Synthetic diiron compounds of the general formula Fe2(µ-S2R)(CO)n(L)6-n (R = alkyl or aromatic groups; L = CN- or phosphines) are versatile models for the active-site cofactor of hydrogen turnover in [FeFe]-hydrogenases. A series of 18 diiron compounds, containing mostly a dithiolate bridge and terminal ligands of increasing complexity, was characterized by X-ray absorption and emission spectroscopy in combination with density functional theory. Fe K-edge absorption and Kß main-line emission spectra revealed the varying geometry and the low-spin state of the Fe(i) centers. Good agreement between experimental and calculated core-to-valence-excitation absorption and radiative valence-to-core-decay emission spectra revealed correlations between spectroscopic and structural features and provided access to the electronic configuration. Four main effects on the diiron core were identified, which were preferentially related to variation either of the dithiolate or of the terminal ligands. Alteration of the dithiolate bridge affected mainly the Fe-Fe bond strength, while more potent donor substitution and ligand field asymmetrization changed the metal charge and valence level localization. In contrast, cyanide ligation altered all relevant properties and, in particular, the frontier molecular orbital energies of the diiron core. Mutual benchmarking of experimental and theoretical parameters provides guidelines to verify the electronic properties of related diiron compounds.

Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases.

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier's principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.