Electron-nuclear double resonance studies of oxidized Escherichia coli sulfite reductase: 1H, 14N, and 57Fe measurements

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the bridged siroheme--[Fe4S4] cluster that forms the catalytically active center of the oxidized hemoprotein subunit (SiRo) of Escherichia coli NADPH-sulfite reductase. The siroheme 57Fe hyperfine coupling (Az = 27.6 MHz, Ay = 26.8 MHz) is similar to that of other high-spin heme systems (A approximately equal to 27 MHz). Bonding parameters obtained from the 14N hyperfine coupling constants of the siroheme pyrrole nitrogens are consistent with a model of a nonplanar pi system of reduced aromaticity. The absence of hyperfine coupling to the 14N of an axial ligand, such as is observed for the histidine 14N of metmyoglobin (Az = 11.55 MHz), rules out the possibility that imidazolate acts as the bridge between the siroheme and the [Fe4S4] cluster. Proton ENDOR of the deuterium-exchanged protein indicates that H2O does not function as a sixth axial ligand and suggests that the ferrisiroheme is five-coordinate. 57Fe ENDOR measurements confirm the results of M?ssbauer spectroscopy for the [Fe4S4] cluster. They also disclose a slight anisotropy of the cluster 57Fe coupling that may be associated with the mechanism by which the siroheme and cluster spins are coupled.     

1H electron-nuclear double resonance of cobalt hemoglobin

Proton 1H electron-nuclear double resonance spectra were obtained from cobalt-substituted hemoglobin A. For oxy cobalt-substituted hemoglobin (CoHb), two major couplings were found. One is exchangeable by deuterium and has a coupling of approximately 5.6 MHz which is assigned to the N epsilon 2-proton of the distal histidine (E7), interacting with the ligand. The other splitting (approximately 2.4-3.2 MHz) is attributed to a methyl-proton on Val (E11). No coupling of protons on the proximal side is resolved. Deoxy CoHb exhibits only one prominent interaction (approximately 1.3 MHz) which is assigned to one of the protons on the proximal histidine (F8). The influence of the distal amino acids His (E7) and Val (E11) on the ligand affinity in oxy CoHb is discussed.     

Superoxidized states of Escherichia coli sulfite reductase heme protein subunit

The oxidized forms of resting and sulfite-complexed Escherichia coli sulfite reductase heme protein subunit react with near-stoichiometric amounts of porphyrexide to produce what is best characterized as a ferrisiroheme pi cation radical. Addition of either sodium ascorbate or NADPH completely regenerates the parent form. Implications of these findings with respect to mechanisms of metal-radical J coupling and catalysis are discussed.     

The heme and Fe4S4 cluster in the crystallographic structure of Escherichia coli sulfite reductase

Isolated hemoprotein subunits of Escherichia coli NADPH:sulfite reductase catalyze the 6-electron reduction of SO2-3 to S2-. The prosthetic groups of the hemoprotein, a siroheme and a Fe4S4 cluster, have been shown by spectroscopy to be tightly coupled. We have crystallized the isolated hemoprotein subunits and produced a 3-A electron density map by x-ray crystallography. A single heavy atom derivative and the native anomalous scattering (from the protein's 5 Fe and several S) were used to calculate the phases. In the electron density map, the cluster has a geometry similar to other Fe4S4 clusters. Both the cluster and the siroheme are found near the surface of the protein. The siroheme and the Fe4S4 cluster pack next to each other in the structure, apparently with a common ligand, consistent with a cysteine S gamma, shared by the siroheme Fe and one of the cluster Fe. The distance from the siroheme Fe to the center of the cluster is 5.5 A and the distance from the siroheme Fe to the nearest cluster Fe is 4.4 A. The edge of the siroheme macrocycle appears to be in Van der Waals contact with a cubane S atom of the cluster. The sixth coordination position of the siroheme Fe appears unoccupied and is quite exposed to the solvent. Some possible implications of the proposed structure on the role of the bridged siroheme-Fe4S4 cluster in catalysis are discussed.     

Activated conformers of Escherichia coli sulfite reductase heme protein subunit

The heme protein subunit of Escherichia coli sulfite reductase shows enhanced reactivity with its substrate and a number of other ligands after a cycle of reduction and reoxidation at alkaline pH. At pH 9.5 this variant of the enzyme possesses at least four EPR-detectable, chloride-sensitive high-spin conformers, in contrast to the single chloride-insensitive species observed in the oxidized, resting enzyme at pH 7.7. Quantitative reversal of the spectral and ligand-binding properties of the "activated" enzyme to those of the resting enzyme is observed on reacidification to pH 7.7. At intermediate pH values, there occurs an acid-catalyzed relaxation of the activated enzyme to the resting form. This reaction is distinct from the one responsible for the accelerated ligand binding and production of multiple EPR conformers, which appears to be regulated by a process with a pK of 8.5.     

Hyperfine evidence of strong double exchange in multimetallic {[Fe4S4]-Fe} active center of Escherichia coli sulfite reductase

 The spin-coupling model of the {[Fe₄S₄]-Fe} center of the active site of sulfite reductase, which includes the Heisenberg exchange and double-exchange interactions, explains the peculiarities of the induced paramagnetism. The effective hyperfine constants of the SiR^–1-NO complex are explained in the model with maximal pair spins S₁₂=S₃₄=9/2 in the ground state formed by strong double exchange. Received: 12 January 1996 / Accepted: 23 January 1996     

A simplifed functional version of the Escherichia coli sulfite reductase

Escherichia coli sulfite reductase (SiR) is a large and soluble enzyme with an alpha(8)beta(4) quaternary structure. Protein alpha (or sulfite reductase flavoprotein) contains both FAD and FMN, whereas protein beta (or sulfite reductase hemoprotein (SiR-HP)) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is set up to arrange the redox cofactors in a FAD-FMN-Fe(4)S(4)-Heme sequence to make an electron pathway between NADPH and sulfite. Whereas alpha spontaneously polymerizes, we have been able to produce SiR-FP60, a monomeric but fully active truncated version of it, lacking the N-terminal part (Zeghouf, M., Fontecave, M., Macherel, D., and Covès, J. (1998) Biochemistry 37, 6114-6123). Here we report the cloning, overproduction, and characterization of the beta subunit. Pure recombinant SiR-HP behaves as a monomer in solution and is identical to the native protein in all its characteristics. Moreover, we demonstrate that the combination of SiR-FP60 and SiR-HP produces a functional 1:1 complex with tight interactions retaining about 20% of the activity of the native SiR. In addition, fully active SiR can be reconstituted by incubation of the octameric sulfite reductase flavoprotein with recombinant SiR-HP. Titration experiments and spectroscopic properties strongly suggest that the holoenzyme should be described as an alpha(8)beta(8) with equal amounts of alpha and beta subunits and that the alpha(8)beta(4) structure is probably not correct.     

31P nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase.

SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Covès, J. (1998) Biochemistry 37, 6117-6123]. SiR-FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.     

Solution structure of the sulfite reductase flavodoxin-like domain from Escherichia coli

The flavoprotein moiety of Escherichia coli sulfite reductase (SiR-FP) is homologous to electron transfer proteins such as cytochrome-P450 reductase (CPR) or nitric oxide synthase (NOS). We report on the three-dimensional structure of SiR-FP18, the flavodoxin-like domain of SiR-FP, which has been determined by NMR. In the holoenzyme, this domain plays an important role by shuttling electrons from the FAD to the hemoprotein (the beta-subunit). The structure presented here was determined using distance and torsion angle information in combination with residual dipolar couplings determined in two different alignment media. Several protein-FMN NOEs allowed us to place the prosthetic group in its binding pocket. The structure is well-resolved, and (15)N relaxation data indicate that SiR-FP18 is a compact domain. The binding interface with cytochrome c, a nonphysiological electron acceptor, has been determined using chemical shift mapping. Comparison of the SiR-FP18 structure with the corresponding domains from CPR and NOS shows that the fold of the protein core is highly conserved, but the analysis of the electrostatic surfaces reveals significant differences between the three domains. These observations are placed in the physiological context so they can contribute to the understanding of the electron transfer mechanism in the SiR holoenzyme.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 1. Siroheme vibrational modes

Resonance Raman (RR) spectra are reported for the hemoprotein subunit (SiR-HP) of Escherichia coli NADPH-sulfite reductase (EC 1.8.1.2) in various ligation and redox states. Comparison of the RR spectra of extracted siroheme and the mu-oxo FeIII dimer of octaethylisobacteriochlorin with those of mu-oxo FeIII octaethylchlorin dimer and mu-oxo FeIII octaethylporphyrin dimer demonstrates that many siroheme bands can be correlated with established porphyrin skeletal modes. Depolarization measurements are a powerful tool in this correlation, since the 45 degrees rotation of the C2 symmetry axis of the isobacteriochlorin ring relative to the chlorin system results in reversal of the polarization properties (polarized vs anomalously polarized) of bands correlating with B1g and B2g modes of porphyrin. Various SiR-HP adducts (CO, NO, CN-, SO3(2-] show upshifted high-frequency bands, characteristic of the low-spin state and consistent with the expected core size sensitivity of the skeletal modes. Fully reduced unliganded SiR-HP (both siroheme and Fe4S4 cluster reduced) in liquid solution displays RR features comparable to those of high-spin ferrous porphyrins; on freezing, the RR spectrum changes, reflecting an apparent mixture of siroheme spin states. At intermediate reduction levels in solution a RR species is observed whose high-frequency bands are upshifted relative to oxidized and fully reduced SiR-HP. This spectrum, thought to arise from the "one-electron" state of SiR-HP (siroheme reduced, cluster oxidized), may be due to S = 1 FeII siroheme.     

1H, 13C and 15N resonance assignments for the oxidized and reduced states of the N-terminal domain of DsbD from Escherichia coli

Viability and pathogenicity of Gram-negative bacteria is linked to the cytochrome c maturation and the oxidative protein folding systems in the periplasm. The transmembrane reductant conductor DsbD is a unique protein which provides the necessary reducing power to both systems through thiol-disulfide exchange reactions in a complex network of protein-protein interactions. The N-terminal domain of DsbD (nDsbD) is the delivery point of the reducing power originating from cytoplasmic thioredoxin to a variety of periplasmic partners. Here we report (1)H, (13)C and (15)N assignments for resonances of nDsbD in its oxidized and reduced states. These assignments provide the starting point for detailed investigations of the interactions of nDsbD with its protein partners.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.     

1H, 13C and 15N backbone and side-chain resonance assignments of reduced CcmG from Escherichia coli

CcmG is a periplasmic, membrane-anchored protein widely distributed in a variety of species. In Escherichia coli, the CcmG protein always acts as a weak reductant in the electron transport chain during cytochrome c maturation (Ccm). Here we report ¹H, ¹⁵N and ¹³C backbone and side-chain resonance assignments of the reduced CcmG protein (residues 19–185, renumbered as 1–167) from E. coli. This work lays the essential basis for the further structural and functional analysis of reduced CcmG.     

Electron paramagnetic resonance and optical evidence for interaction between siroheme and Fe4S4 prosthetic groups in complexes of Escherichia coli sulfite reductase hemoprotein with added ligands

Janick & Siegel [Janick, P. A., & Siegel, L. M. (1982) Biochemistry 21, 3538-3547] showed that the EPR spectrum of the reduced Fe4S4 center (S = 1/2) in fully reduced native ("unligated") Escherichia coli NADPH-sulfite reductase hemoprotein subunit (SiR-HP) is perturbed by interaction with paramagnetic ferrous siroheme (S = 1 or 2) to yield several novel sets of EPR signals: one set with all g values between 2.0 and 2.8, termed "S = 1/2" type, and two sets with the lowest field g value between 4.7 and 5.4, termed "S = 3/2" type. The present study has shown that EPR spectra of fully reduced SiR-HP are nearly quantitatively converted to the classical "g = 1.94" type typical of S = 1/2 Fe4S4 clusters when the heme has been ligated by strong field ligands such as CO, CN-, S2-, and AsO2-, converting the ferroheme to S = 0. However, the exact line shapes and g values of the g = 1.94 differ markedly when different ligands are bound to the heme. Also, optical difference spectra taken between enzyme species in which the heme is kept in the same (Fe2+) oxidation state while the Fe4S4 center is reduced or oxidized show that the optical spectrum of the ligated siroheme is sensitive to the oxidation state of the Fe4S4 cluster. These results indicate that the heme-Fe4S4 interaction of native SiR-HP persists even when the heme Fe is bound to exogenous ligands. We have also found that the g values of the exchange-coupled S = 1/2 and S V 3/2 type signals of native reduced SiR-HP can be significantly shifted by addition of potential weak field heme ligands--halides and formate--or low concentrations of certain chaotropic agents--guanidinium salts and dimethyl sulfoxide--to the fully reduced enzyme. Such agents can also promote interconversion of the S = 1/2 and S = 3/2 type signals. These effects are reversed on removal of the agent. Treatment of reduced SiR-HP with relatively large concentrations of chaotropes, e.g., 60% dimethyl sulfoxide or 2 or 3 M urea, leads to abolition of the S = 1/2 and S = 3/2 EPR signals and their replacement by signals of the g = 1.94 type.     

Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide

Kemp, John D. (University of California, Los Angeles), and Daniel E. Atkinson. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide. J. Bacteriol. 92:628-634. 1966.-A nitrite reductase specific for reduced nicotinamide adenine dinucleotide (NADH(2)) appears to be responsible for in vivo nitrite reduction by Escherichia coli strain Bn. In extracts, the reduction product is ammonium, and the ratio of NADH(2) oxidized to nitrite reduced or to ammonium produced is 3. The Michaelis constant for nitrite is 10 mum. The enzyme is induced by nitrite, and the ability of intact cells to reduce nitrite parallels the level of NADH(2)-specific nitrite reductase activity demonstrable in cell-free preparations. Crude extracts of strain Bn will also reduce hydroxylamine, but not nitrate or sulfite, at the expense of NADH(2). Kinetic observations indicate that hydroxylamine and nitrite may both be reduced at the same active site. The high apparent Michaelis constant for hydroxylamine (1.5 mm), however, seems to exclude hydroxylamine as an intermediate in nitrite reduction. In vitro activity is enhanced by preincubation with nitrite, and decreased by preincubation with NADH(2).     

Reactivity, secondary structure, and molecular topology of the Escherichia coli sulfite reductase flavodoxin-like domain

The flavodoxin-like domain, missing in the three-dimensional structure of the monomeric, simplified model of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has now been expressed independently. This 168 amino acid protein was named SiR-FP18 with respect to its native molecular weight and represents the FMN-binding domain of SiR-FP. This simplified biological object has kept the main characteristics of its counterpart in the native protein. It could incorporate FMN exclusively and stabilize a neutral air-stable semiquinone radical. Both the radical and the fully reduced forms of SiR-FP18 were able to transfer their electrons to DCPIP or cytochrome c quantitatively. SiR-FP18 was able to form a highly stable complex with SiR-HP, the hemoprotein component of the sulfite reductase containing an iron-sulfur cluster coupled to a siroheme. In agreement with the postulated catalytic cycle of SiR-FP, only the fully reduced form of SiR-FP18 could transfer one electron to SiR-HP, the transferred electron being localized exclusively on the heme. As isolated SiR-FP18 has kept the main characteristics of the FMN-binding domain of the native protein, a structural analysis by NMR was performed in order to complete the partial structure obtained previously. Structural modeling was performed using sequence homologues, cytochrome P450 reductase (CPR; 29% identity) and bacterial cytochrome P450 (P450-BM3; 26% identity), as conformational templates. These sequences were anchored using common secondary structural elements identified from heteronuclear NMR data measured on the protein backbone. The resulting structural model was validated, and subsequently refined using residual (C(alpha)-C', N-H(N), and C'-H(N)) dipolar couplings measured in an anisotropic medium. The overall fold of SiR-FP18 is very similar to that of bacterial flavodoxins and of the flavodoxin-like domain in CPR or P450-BM3.