Correct assembly of iron-sulfur cluster FS0 into Escherichia coli dimethyl sulfoxide reductase (DmsABC) is a prerequisite for molybdenum cofactor insertion

The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ～ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of dimethyl sulfoxide reduction. Comparison with nitrate reductase A (NarGHI) indicates that a sequence of residues ((18)CTVNC(22)) plays a role in both FS0 and Mo-bisPGD coordination. A DmsA(ΔN21) mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from (18)C(A)TVNC(B)GSRC(C)P(27) to (18)C(A)TYC(B)GVGC(C)G(26) (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsA(R61K) mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.     

The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state

We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). FS0 is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.     

A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase

We have investigated the final steps of complex iron–sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron–sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability. DmsA-Cys59 is a ligand to the FS0 [4Fe–4S] cluster. In the presence of trace amounts of molybdate, the Cys59Ser variant assembles normally to the cytoplasmic membrane and supports respiratory growth on DMSO, although the ground state of FS0 as determined by EPR is converted from high-spin (S = 3/2) to low-spin (S = 1/2). In the presence of the molybdenum antagonist tungstate, wild-type DmsABC lacks Mo-bisPGD, but is translocated via the Tat translocon and assembles on the periplasmic side of the membrane as an apoenzyme. The Cys59Ser variant cannot overcome the dual insults of amino acid substitution plus lack of Mo-bisPGD, leading to degradation of the DmsABC subunits. This indicates that the cofactor can serve as a chemical chaperone to mitigate the destabilizing effects of alteration of the FS0 cluster. These results provide insights into the role of the Mo–bisPGD–protein interaction in stabilizing the tertiary structure of DmsA during enzyme maturation.     

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser⁷¹⁹, NarG-His¹¹⁶³, and NarG-His¹¹⁸⁴); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His¹⁰⁹² and NarG-His¹⁰⁹⁸). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔE_m values of −88 and −36 mV, respectively). Ala variants of His¹⁰⁹² and His¹⁰⁹⁸ also elicit large ΔE_m values of −143 and −101 mV, respectively. An Arg variant of His¹⁰⁹² elicits a small ΔE_m of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum E_m value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.     

The Small Subunit AroB of Arsenite Oxidase: LESSONS ON THE [2Fe-2S] RIESKE PROTEIN SUPERFAMILY

Here, we describe the characterization of the [2Fe-2S] clusters of arsenite oxidases from Rhizobium sp. NT-26 and Ralstonia sp. 22. Both reduced Rieske proteins feature EPR signals similar to their homologs from Rieske-cyt b complexes, with g values at 2.027, 1.88, and 1.77. Redox titrations in a range of pH values showed that both [2Fe-2S] centers have constant E_m values up to pH 8 at ∼+210 mV. Above this pH value, the E_m values of both centers are pH-dependent, similar to what is observed for the Rieske-cyt b complexes. The redox properties of these two proteins, together with the low E_m value (+160 mV) of the Alcaligenes faecalis arsenite oxidase Rieske (confirmed herein), are in line with the structural determinants observed in the primary sequences, which have previously been deduced from the study of Rieske-cyt b complexes. Since the published E_m value of the Chloroflexus aurantiacus Rieske (+100 mV) is in conflict with this sequence analysis, we re-analyzed membrane samples of this organism and obtain a new value (+200 mV). Arsenite oxidase activity was affected by quinols and quinol analogs, which is similar to what is found with the Rieske-cyt b complexes. Together, these results show that the Rieske protein of arsenite oxidase shares numerous properties with its counterpart in the Rieske-cyt b complex. However, two cysteine residues, strictly conserved in the Rieske-cyt b-Rieske and considered to be crucial for its function, are not conserved in the arsenite oxidase counterpart. We discuss the role of these residues.     

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q_D) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q_D site (MSQ_D) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with ¹H₂O/²H₂O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ_D binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in ¹H₂O and ²H₂O samples and by selective detection of the exchanged deuterons using Q-band ²H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b_D. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q_D site are discussed, in light of the unusually high thermodynamic stability of MSQ_D.     

Modifications of Protein Environment of the [2Fe-2S] Cluster of the bc1 Complex: EFFECTS ON THE BIOPHYSICAL PROPERTIES OF THE RIESKE IRON-SULFUR PROTEIN AND ON THE KINETICS OF THE COMPLEX*

The rate-determining step in the overall turnover of the bc₁ complex is electron transfer from ubiquinol to the Rieske iron-sulfur protein (ISP) at the Q_o-site. Structures of the ISP from Rhodobacter sphaeroides show that serine 154 and tyrosine 156 form H-bonds to S-1 of the [2Fe-2S] cluster and to the sulfur atom of the cysteine liganding Fe-1 of the cluster, respectively. These are responsible in part for the high potential (E_m,7 ∼300 mV) and low pK_a (7.6) of the ISP, which determine the overall reaction rate of the bc₁ complex. We have made site-directed mutations at these residues, measured thermodynamic properties using protein film voltammetry to evaluate the E_m and pK_a values of ISPs, explored the local proton environment through two-dimensional electron spin echo envelope modulation, and characterized function in strains S154T, S154C, S154A, Y156F, and Y156W. Alterations in reaction rate were investigated under conditions in which concentration of one substrate (ubiquinol or ISP_ox) was saturating and the other was varied, allowing calculation of kinetic terms and relative affinities. These studies confirm that H-bonds to the cluster or its ligands are important determinants of the electrochemical characteristics of the ISP, likely through electron affinity of the interacting atom and the geometry of the H-bonding neighborhood. The calculated parameters were used in a detailed Marcus-Brønsted analysis of the dependence of rate on driving force and pH. The proton-first-then-electron model proposed accounts naturally for the effects of mutation on the overall reaction.     

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E_m) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys⁻, and active site His/CysS⁻-ligated heme, respectively. Exposing SoxAX to S₂O₄²⁻, a substrate analog with E_m ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E_m ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS⁻) ligand to the active site heme. This provides the first evidence for the dissociation of CysS⁻ that has been proposed as a key event in SoxAX catalysis.     

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

The membrane-bound high-molecular-mass cytochromes c from Desulfovibrio gigas and Desulfovibrio vulgaris Hildenborough; EPR and Mössbauer studies

The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E ⁰s of –30?mV to –100?mV, three to four hemes have E ⁰s around –170?mV, and seven to eight hemes have a lower E ⁰ of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow.     

Environment of TyrZ in photosystem II from Thermosynechococcus elongatus in which PsbA2 is the D1 protein

The main cofactors that determine the photosystem II (PSII) oxygen evolution activity are borne by the D1 and D2 subunits. In the cyanobacterium Thermosynechococcus elongatus, there are three psbA genes coding for D1. Among the 344 residues constituting D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. Here, we present the first study of PsbA2-PSII. Using EPR and UV-visible time-resolved absorption spectroscopy, we show that: (i) the time-resolved EPR spectrum of Tyr_Z^• in the (S₃Tyr_Z^•)′ is slightly modified; (ii) the split EPR signal arising from Tyr_Z^• in the (S₂Tyr_Z^•)′ state induced by near-infrared illumination at 4.2 K of the S₃Tyr_Z state is significantly modified; and (iii) the slow phases of P₆₈₀^+⋅ reduction by Tyr_Z are slowed down from the hundreds of μs time range to the ms time range, whereas both the S₁Tyr_Z^• → S₂Tyr_Z and the S₃Tyr_Z^• → S₀Tyr_Z + O₂ transition kinetics remained similar to those in PsbA(1/3)-PSII. These results show that the geometry of the Tyr_Z phenol and its environment, likely the Tyr-O···H···Nϵ-His bonding, are modified in PsbA2-PSII when compared with PsbA(1/3)-PSII. They also point to the dynamics of the proton-coupled electron transfer processes associated with the oxidation of Tyr_Z being affected. From sequence comparison, we propose that the C144P and P173M substitutions in PsbA2-PSII versus PsbA(1/3)-PSII, respectively located upstream of the α-helix bearing Tyr_Z and between the two α-helices bearing Tyr_Z and its hydrogen-bonded partner, His-190, are responsible for these changes.     

Thermostabilization of ovalbumin by alkaline treatment: Examination of the possible roles of d-serine residues

It was revealed from the crystal structure analysis of S-ovalbumin (S-OVA) formed by alkaline treatment that Ser164, Ser236, and Ser320 take the d-amino acid residue configuration (Yamasaki et al., J Biol Chem 2003; 278:35524–35530). To address the implications of a d-configuration for these Ser residues in S-OVA formation, three mutant OVAs (S164A, S236A, and S320A) were generated to compare their thermostabilities before and after alkaline treatment. Following alkaline treatment, S236A showed a marked increase in melting temperature similar to the wild type (ΔT_m, +9°C) which corresponded to the formation of S-OVA, whereas the increment in T_m for both S164A and S320A was only 4.5°C. Furthermore, the T_m value of the double mutant S164/320A remained unchanged after alkaline treatment, supporting the relevance of Ser164 and Ser320 for thermostabilization of OVA. As Arg142 was predicted to interact with D-Ser164 upon S-OVA formation, it was substituted to Ala to generate R142A. The resulting increment in T_m of mutant R142A after alkaline treatment was 5.8°C. The double mutant R142/S320A was therefore prepared to eliminate the participation of Ser320 in thermostabilization, and its T_m value was compared before and after alkaline treatment. As expected, the increase in T_m for the double mutant was only 1.2°C. Taken together, the data suggest that d-configuration of Ser164 caused by alkaline treatment favors interaction with Arg142 through conformational changes of the side chain. These results strongly supported the participation of the configurational inversion of both Ser164 and Ser320 residues in the formation of S-OVA.     

Studies of the function of the membrane-bound iron-sulfur centers of the photosynthetic bacterium Chromatium vinosum

Chromatophores from the photosynthetic bacterium, Chromatium vinosum, have been prepared which photoreduce NAD⁺ with either succinate or reduced dichlorophenolindophenol as electron donors. NAD⁺ reduction is inhibited by uncouplers as well as inhibitors of cyclic photophosphorylation. These chromatophores contain several bound iron-sulfur centers which have been detected by low-temperature EPR spectroscopy. One center, having a g 2.01 EPR signal in the oxidized state, has E_m7.5 = +50 mV and is partially reduced by succinate in the dark. Three iron-sulfur centers having g 1.93 EPR signals have been resolved by redox titration, and the E_m7.5 values of these centers are ?50, ?175 and ?250 mV, respectively. Studies of the involvement of these centers in electron transfer from donors to NAD⁺ have indicated that the center with E_m = ?50 mV is succinate reducible in the dark and appears to be analogous to center S-1 of succinic dehydrogenase in other systems. An additional g 1.93 iron-sulfur center can be photoreduced in the presence of electron donors and this reduction is inhibited by uncouplers. The possible role of the two low-potential iron-sulfur centers in relation to the dehydrogenases functioning in NAD⁺ reduction is considered.     

A relationship between the midpoint potential of the primary acceptor and low temperature photochemistry in photosystem II

Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (E_m ? ?50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (E_m ? ?275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the E_m-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.     

Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

The functional role of cytochrome (cyt) b₅₅₉ in photosystem II (PSII) was investigated in H22Kα and Y18Sα cyt b₅₅₉ mutants of the cyanobacterium Synechocystis sp. PCC6803. H22Kα and Y18Sα cyt b₅₅₉ mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b₅₅₉ in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b₅₅₉ in H22Kα mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b₅₅₉ in Y18Sα mutant PSII core complexes was shifted to 561 nm. Y18Sα and H22Kα mutant PSII core complexes contained predominately the low potential form of cyt b₅₅₉. The findings lend support to the concept that the redox properties of cyt b₅₅₉ are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b₅₅₉ mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b₅₅₉ in protection of PSII under photoinhibition conditions in vivo.     

Diversity of Cytochrome bc Complexes: Example of the Rieske Protein in Green Sulfur Bacteria

The Rieske 2Fe2S cluster of Chlorobium limicola forma thiosulfatophilum strain tassajara was studied by electron paramagnetic resonance spectroscopy. Two distinct orientations of its g tensor were observed in oriented samples corresponding to differing conformations of the protein. Only one of the two conformations persisted after treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. A redox midpoint potential (E_m) of +160 mV in the pH range of 6 to 7.7 and a decreasing E_m (−60 to −80 mV/pH unit) above pH 7.7 were found. The implications of the existence of differing conformational states of the Rieske protein, as well as of the shape of its E_m-versus-pH curve, in green sulfur bacteria are discussed.     

Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis

Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe₄S₄]^2+,+ cluster (E _m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and W(V)/W(VI) states at physiologically relevant potentials (E _m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na₂S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E _m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E _m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR. Received: 9 September 1999 / Accepted: 17 February 2000     

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2 + Entry

Store-operated Ca^2 + entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca^2 + entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca^2 + release-activated Ca^2 + channel current (I_CRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and I_CRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca²⁺ depletion-dependent oligomerization of the luminal Ca^2 +-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.