Mitochondrial targeting sequence of magnetoreceptor MagR: More than just targeting.

Iron-sulfur clusters are essential cofactors for proteins involved in various biological processes, such as electron transport, biosynthetic reactions, DNA repair, and gene expression regulation. Iron-sulfur cluster assembly protein IscA1 (or MagR) is found within the mitochondria of most eukaryotes. Magnetoreceptor (MagR) is a highly conserved A-type iron and iron-sulfur cluster-binding protein, characterized by two distinct types of iron-sulfur clusters, [2Fe-2S] and [3Fe-4S], each conferring unique magnetic properties. MagR forms a rod-like polymer structure in complex with photoreceptive cryptochrome (Cry) and serves as a putative magnetoreceptor for retrieving geomagnetic information in animal navigation. Although the N-terminal sequences of MagR vary among species, their specific function remains unknown. In the present study, we found that the N-terminal sequences of pigeon MagR, previously thought to serve as a mitochondrial targeting signal (MTS), were not cleaved following mitochondrial entry but instead modulated the efficiency with which iron-sulfur clusters and irons are bound. Moreover, the N-terminal region of MagR was required for the formation of a stable MagR/Cry complex. Thus, the N-terminal sequences in pigeon MagR fulfil more important functional roles than just mitochondrial targeting. These results further extend our understanding of the function of MagR and provide new insights into the origin of magnetoreception from an evolutionary perspective.

Multimodal Spectroscopic Analysis of the Fe-S Clusters of the as-Isolated Escherichia coli SufBC2D Complex.

Iron-sulfur (Fe-S) clusters are essential inorganic cofactors dedicated to a wide range of biological functions, including electron transfer and catalysis. Specialized multiprotein machineries present in all types of organisms support their biosynthesis. These machineries encompass a scaffold protein, on which Fe-S clusters are assembled before being transferred to cellular targets. Here, we describe the first characterization of the native Fe-S cluster of the anaerobically purified SufBC2D scaffold from Escherichia coli by XAS and Mössbauer, UV-visible absorption, and EPR spectroscopies. Interestingly, we propose that SufBC2D harbors two iron-sulfur-containing species, a [2Fe-2S] cluster and an as-yet unidentified species. Mutagenesis and biochemistry were used to propose amino acid ligands for the [2Fe-2S] cluster, supporting the hypothesis that both SufB and SufD are involved in the Fe-S cluster ligation. The [2Fe-2S] cluster can be transferred to ferredoxin in agreement with the SufBC2D scaffold function. These results are discussed in the context of Fe-S cluster biogenesis.

Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

H2 production under stress: [FeFe]­hydrogenases reveal strong stability in high pressure environments.

Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of H2 into protons and electrons and the reverse reaction. A subgroup is formed by the [FeFe]­hydrogenases, which are the most efficient enzymes of microbes for catalytic H2 conversion. We have determined the stability and activity of two [FeFe]­hydrogenases under high temperature and pressure conditions employing FTIR spectroscopy and the high-pressure stopped-flow methodology in combination with fast UV/Vis detection. Our data show high temperature stability and an increase in activity up to the unfolding temperatures of the enzymes. Remarkably, both enzymes reveal a very high pressure stability of their structure, even up to pressures of several kbars. Their high pressure-stability enables high enzymatic activity up to 2 kbar, which largely exceeds the pressure limit encountered by organisms in the deep sea and sub-seafloor on Earth.

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.

Biochemical and cellular characterization of the CISD3 protein: Molecular bases of cluster release and destabilizing effects of nitric oxide.

The NEET proteins, an important family of iron-sulfur (Fe-S) proteins, have generated a strong interest due to their involvement in diverse diseases such as cancer, diabetes, and neurodegenerative disorders. Among the human NEET proteins, CISD3 has been the least studied, and its functional role is still largely unknown. We have investigated the biochemical features of CISD3 at the atomic and in cellulo levels upon challenge with different stress conditions i.e., iron deficiency, exposure to hydrogen peroxide, and nitric oxide. The redox and cellular stability properties of the protein agree on a predominance of reduced form of CISD3 in the cells. Upon the addition of iron chelators, CISD3 loses its Fe-S clusters and becomes unstructured, and its cellular level drastically decreases. Chemical shift perturbation measurements suggest that, upon cluster oxidation, the protein undergoes a conformational change at the C-terminal CDGSH domain, which determines the instability of the oxidized state. This redox-associated conformational change may be the source of cooperative electron transfer via the two [Fe2S2] clusters in CISD3, which displays a single sharp voltammetric signal at -31 mV versus SHE. Oxidized CISD3 is particularly sensitive to the presence of hydrogen peroxide in vitro, whereas only the reduced form is able to bind nitric oxide. Paramagnetic NMR provides clear evidence that, upon NO binding, the cluster is disassembled but iron ions are still bound to the protein. Accordingly, in cellulo CISD3 is unaffected by oxidative stress induced by hydrogen peroxide but it becomes highly unstable in response to nitric oxide treatment.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.

Kinetic Modeling of the Reversible or Irreversible Electrochemical Responses of FeFe-Hydrogenases.

The enzyme FeFe-hydrogenase catalyzes H2 evolution and oxidation at an active site that consists of a [4Fe-4S] cluster bridged to a [Fe2(CO)3(CN)2(azadithiolate)] subsite. Previous investigations of its mechanism were mostly conducted on a few "prototypical" FeFe-hydrogenases, such as that from Chlamydomonas reinhardtii(Cr HydA1), but atypical hydrogenases have recently been characterized in an effort to explore the diversity of this class of enzymes. We aim at understanding why prototypical hydrogenases are active in either direction of the reaction in response to a small deviation from equilibrium, whereas the homologous enzyme from Thermoanaerobacter mathranii (Tam HydS) shows activity only under conditions of very high driving force, a behavior that was referred to as "irreversible catalysis". We follow up on previous spectroscopic studies and recent developments in the kinetic modeling of bidirectional reactions to investigate and compare the catalytic cycles of Cr HydA1 and Tam HydS under conditions of direct electron transfer with an electrode. We compare the hypothetical catalytic cycles described in the literature, and we show that the observed changes in catalytic activity as a function of potential, pH, and H2 concentration can be explained with the assumption that the same catalytic mechanism applies. This helps us identify which variations in properties of the catalytic intermediates give rise to the distinct "reversible" or "irreversible" catalytic behaviors.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

The Active-Site [4Fe-4S] Cluster in the Isoprenoid Biosynthesis Enzyme IspH Adopts Unexpected Redox States during Ligand Binding and Catalysis.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase, or IspH (formerly known as LytB), catalyzes the terminal step of the bacterial methylerythritol phosphate (MEP) pathway for isoprene synthesis. This step converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into one of two possible isomeric products, either isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). This reaction involves the removal of the C4 hydroxyl group of HMBPP and addition of two electrons. IspH contains a [4Fe-4S] cluster in its active site, and multiple cluster-based paramagnetic species of uncertain redox and ligation states can be detected after incubation with reductant, addition of a ligand, or during catalysis. To characterize the clusters in these species, 57Fe-labeled samples of IspH were prepared and studied by electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies. Notably, this ENDOR study provides a rarely reported, complete determination of the 57Fe hyperfine tensors for all four Fe ions in a [4Fe-4S] cluster. The resting state of the enzyme (Ox) has a diamagnetic [4Fe-4S]2+ cluster. Reduction generates [4Fe-4S]+ (Red) with both S = 1/2 and S = 3/2 spin ground states. When the reduced enzyme is incubated with substrate, a transient paramagnetic reaction intermediate is detected (Int) which is thought to contain a cluster-bound substrate-derived species. The EPR properties of Int are indicative of a 3+ iron-sulfur cluster oxidation state, and the Mössbauer spectra presented here confirm this. Incubation of reduced enzyme with the product IPP induced yet another paramagnetic [4Fe-4S]+ species (Red+P) with S = 1/2. However, the g-tensor of this state is commonly associated with a 3+ oxidation state, while Mössbauer parameters show features typical for 2+ clusters. Implications of these complicated results are discussed.

Ferredoxin 1 is essential for embryonic development and lipid homeostasis.

Mammalian ferredoxin 1 and 2 (FDX1/2) belong to an evolutionary conserved family of iron-sulfur cluster containing proteins and act as electron shutters between ferredoxin reductase (FDXR) and numerous proteins involved in critical biological pathways. FDX1 is involved in biogenesis of steroids and bile acids, Vitamin A/D metabolism, and lipoylation of tricarboxylic acid (TCA) cycle enzymes. FDX1 has been extensively characterized biochemically but its role in physiology and lipid metabolism has not been explored. In this study, we generated Fdx1-deficient mice and showed that knockout of both alleles of the Fdx1 gene led to embryonic lethality. We also showed that like Fdxr+/-+/-, Fdx1+/-+/- had a shorter life span and were prone to steatohepatitis. However, unlike Fdxr+/-+/-, Fdx1+/-+/- were not prone to spontaneous tumors. Additionally, we showed that FDX1 deficiency led to lipid droplet accumulation possibly via the ABCA1-SREBP1/2 pathway. Specifically, untargeted lipidomic analysis showed that FDX1 deficiency led to alterations in several classes of lipids, including cholesterol, triacylglycerides, acylcarnitines, ceramides, phospholipids and lysophospholipids. Taken together, our data indicate that FDX1 is essential for mammalian embryonic development and lipid homeostasis at both cellular and organismal levels.

Probing the mechanism of the dedicated NO sensor [4Fe-4S] NsrR: the effect of cluster ligand environment.

NsrR from Streptomyces coelicolor is a bacterial nitric oxide (NO) sensor/nitrosative stress regulator as its primary function, and has been shown to have differential response at low, mid, and high levels of NO. These must correspond to discrete structural changes at the protein-bound [4Fe-4S] cluster in response to stepwise nitrosylation of the cluster. We have investigated the effect of the monohapto carboxylate ligand in the site differentiated [4Fe-4S] cluster cofactor of the protein NsrR on modulating its reactivity to NO with a focus on indentifying mechanistic intermediates. We have prepared a synthetic model [4Fe-4S] cluster complex with tripodal ligand and one single site differentiated site occupied by either thiolate or carboxylate ligand. We report here the mechanistic details of sequential steps of nitrosylation as observed by ESI MS and IR spectroscopy. Parallel non-denaturing mass spectrometry analyses were performed using site-differentiated variants of NsrR with the native aspartic acid, cysteine, or alanine in the position of the forth ligand to the cluster. A mono-nitrosylated synthetic [4Fe-4S] cluster was observed for the first time in a biologically-relevant thiolate-based coordination environment. Combined synthetic and protein data give unprecedented clarity in the modulation of nitrosylation of a [4Fe-4S] cluster.

A Dynamic Water Channel Affects O2 Stability in [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4FeH ) and a unique diiron moiety (2FeH ). Previous attempts to prevent O2 -induced damage have focused on enhancing the protein's sieving effect for O2 by blocking the hydrophobic gas channels that connect the protein surface and the 2FeH . In this study, we aimed to block an O2 diffusion pathway and shield 4FeH instead. Molecular dynamics (MD) simulations identified a novel water channel (WH ) surrounding the H-cluster. As this hydrophilic path may be accessible for O2 molecules we applied site-directed mutagenesis targeting amino acids along WH in proximity to 4FeH to block O2 diffusion. Protein film electrochemistry experiments demonstrate increased O2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O2 in the environment of the 4FeH in both cases. The results strongly suggest that, in wild type proteins, O2 diffuses from the 4FeH to the 2FeH . These results reveal new strategies for improving the O2 stability of [FeFe]-hydrogenases by focusing on the O2 diffusion network near the active site.

Structure-based insights into the mechanism of [4Fe-4S]-dependent sulfur insertase LarE.

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP-dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe-4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster-free (apo-) and cluster-bound (holo-) forms. The structure of holo-MmLarE shows that the [4Fe-4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein-bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo-MmLarE and the previously reported biochemical investigations of holo-LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe-5S] intermediate, and transfers it to the substrate, as in the case of [4Fe-4S]-dependent tRNA thiolation enzymes.

Acyl and CO Ligands in the [Fe]-Hydrogenase Cofactor Scramble upon Photolysis.

[Fe]-hydrogenase harbors the iron-guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl-carbon, pyridinol-nitrogen, cysteine-thiolate and two CO as ligands. Irradiation with UV-A/blue light decomposes the FeGP cofactor to a 6-carboxymethyl-4-guanylyl-2-pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl- and CO-ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18 O1 -carboxy]-group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18 O-labeled FeGP cofactor revealed that the produced [18 O1 ]-acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time-resolved infrared spectroscopy indicated that an acyl-Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light-excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2 -activating natural complex.

C-Cl Bond Activation at Rotated vs Unrotated Dinuclear Site Related to [FeFe]-Hydrogenases.

The novel dinuclear complex related to the [FeFe]-hydrogenases active site, [Fe2(µ-pdt)(κ2-dmpe)2(CO)2] (1), is highly reactive toward chlorinated compounds CHxCl4-x (x = 1, 2) affording selectively terminal or bridging chloro diiron isomers through a C-Cl bond activation. DFT calculations suggest a cooperative mechanism involving a formal concerted regioselective chloronium transfer depending on the unrotated or rotated conformation of two isomers of 1.

Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway.

Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed.

Maturation of the [FeFe]-Hydrogenase: Direct Transfer of the (κ3 -cysteinate)FeII (CN)(CO)2 Complex B from HydG to HydE.

[FeFe]-hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H-cluster, combining a [Fe4 S4 ] center with a binuclear iron center ([2Fe]H ). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L-tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H -subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X-ray crystallography, we successfully determined its structure at 1.3 Šresolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post-synthesis, highlighting a transient interaction between the two proteins.

Insights into the Molecular Mechanism of Formaldehyde Inhibition of [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are efficient H2 converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover. Kinetic assays via protein film electrochemistry show the CpI variant C299D is significantly less inhibited by HCHO, corroborating the structural results. By combining our data from protein crystallography, site-directed mutagenesis and protein film electrochemistry, a reaction mechanism involving the cofactor's amine base, the thiol group of C299 and HCHO can be deduced. In addition to the specific case of [FeFe]-hydrogenases, our study provides additional insights into the reactions between HCHO and protein molecules.

Virtual Model Compound Approach for Calculating Redox Potentials of [Fe2S2]-Cys4 Centers in Proteins - Structure Quality Matters.

The midpoint potential of the [Fe2S2]-Cys4-cluster in proteins is known to vary between -200 and -450 mV. This variation is caused by the different electrostatic environment of the cluster in the respective proteins. Continuum electrostatics can quantify the impact of the protein environment on the redox potential. Thus, if the redox potential of a [Fe2S2]-Cys4-cluster model compound in aqueous solution would be known, then redox potentials in various protein complexes could be calculated. However, [Fe2S2]-Cys4-cluster models are not water-soluble, and thus, their redox potential can not be measured in aqueous solution. To overcome this problem, we introduce a method that we call Virtual Model Compound Approach (VMCA) to extrapolate the model redox potential from known redox potentials of proteins. We carefully selected high-resolution structures for our analysis and divide them into a fit set, for fitting the model redox potential, and an independent test set, to check the validity of the model redox potential. However, from our analysis, we realized that the some structures can not be used as downloaded from the PDB but had to be re-refined in order to calculate reliable redox potentials. Because of the re-refinement, we were able to significantly reduce the standard deviation of our derived model redox potential for the [Fe2S2]-Cys4-cluster from 31 mV to 10 mV. As the model redox potential, we obtained -184 mV. This model redox potential can be used to analyze the redox behavior of [Fe2S2]-Cys4-clusters in larger protein complexes.