Ming-Yih Liu

STUDIES ON THE REDOX CENTERS OF THE TERMINAL OXIDASE FROM DESULFOVIBRIO GIGAS AND EVIDENCE FOR ITS INTERACTION WITH RUBREDOXIN

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducerDesulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at ∼2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 ± 15 mV for the quinone/semiquinone couple and at −130 ± 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is −350 ± 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Purification and Characterization of an Iron Superoxide Dismutase and a Catalase from the Sulfate-Reducing Bacterium Desulfovibrio gigas

The iron-containing superoxide dismutase (FeSOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) enzymes constitutively expressed by the strictly anaerobic bacterium Desulfovibrio gigas were purified and characterized. The FeSOD, isolated as a homodimer of 22-kDa subunits, has a specific activity of 1,900 U/mg and exhibits an electron paramagnetic resonance (EPR) spectrum characteristic of high-spin ferric iron in a rhombically distorted ligand field. Like other FeSODs from different organisms, D. gigas FeSOD is sensitive to H(2)O(2) and azide but not to cyanide. The N-terminal amino acid sequence shows a high degree of homology with other SODs from different sources. On the other hand, D. gigas catalase has an estimated molecular mass of 186 +/- 8 kDa, consisting of three subunits of 61 kDa, and shows no peroxidase activity. This enzyme is very sensitive to H(2)O(2) and cyanide and only slightly sensitive to sulfide. The native enzyme contains one heme per molecule and exhibits a characteristic high-spin ferric-heme EPR spectrum (g(y,x) = 6.4, 5.4); it has a specific activity of 4,200 U/mg, which is unusually low for this class of enzyme. The importance of these two enzymes in the context of oxygen utilization by this anaerobic organism is discussed.

Crystal structure of a secondary vitamin D3 binding site of milk β-lactoglobulin

β‐lactoglobulin (β‐LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly β structure. The structural function of the only α‐helix with three turns at the C‐terminus is unknown. Vitamin D3 binds to the central calyx formed by the β‐strands. Whether there are two vitamin D binding‐sites in each β‐LG molecule has been a subject of controversy. Here, we report a second vitamin D3 binding site identified by synchrotron X‐ray diffraction (at 2.4 Å resolution). In the central calyx binding mode, the aliphatic tail of vitamin D3 clearly inserts into the binding cavity, where the 3‐OH group of vitamin D3 binds externally. The electron density map suggests that the 3‐OH group interacts with the carbonyl of Lys‐60 forming a hydrogen bond (2.97 Å). The second binding site, however, is near the surface at the C‐terminus (residues 136–149) containing part of an α‐helix and a β‐strand I with 17.91 Å in length, while the span of vitamin D3 is about 12.51 Å. A remarkable feature of the second exosite is that it combines an amphipathic α‐helix providing nonpolar residues (Phe‐136, Ala‐139, and Leu‐140) and a β‐strand providing a nonpolar (Ile‐147) and a buried polar residue (Arg‐148). They are linked by a hydrophobic loop (Ala‐142, Leu‐143, Pro‐144, and Met‐145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D3 binding. This finding provides a new insight into the interaction between vitamin D3 and β‐LG, in which the exosite may provide another route for the transport of vitamin D3 in vitamin D3 fortified dairy products. Atomic coordinates for the crystal structure of β‐LG‐vitamin D3 complex described in this work have been deposited in the PDB (access code 2GJ5). Proteins 2008.

Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)

Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT (Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the Ki value for dehydroepiandrosterone (DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans.

A bacterioferritin was recently isolated from the anaerobic sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841–6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron‐coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate‐reducing bacteria.

Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr‐I and Dsr‐II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α2β2γ2 structure of Dsr‐I contains eight [4Fe–4S] clusters, two saddle‐shaped sirohaems and two flat sirohydrochlorins. In Dsr‐II, the [4Fe–4S] cluster associated with the sirohaem in Dsr‐I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr‐I and Dsr‐II confirm the co‐factor structures, whereas EPR of a third but inactive form, Dsr‐III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr‐I and Dsr‐II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ‐subunit C‐terminus is inserted into a positively charged channel formed between the α‐ and β‐subunits, with its conserved terminal Cysγ104 side‐chain covalently linked to the CHA atom of the sirohaem in Dsr‐I. In Dsr‐II, the thioether bond is broken, and the Cysγ104 side‐chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S3O62−, S2O32− and S2−.

On the nature of the oxidation-reduction properties of nitrite reductase from Desulfovibriodesulfuricans

Abstract Nitrite reductase as isolated from Desulfovibrio desulfuricans shows a complex set of rhombically distorted high-spin ferric heme and low-spin ferric heme resonances at 11oK. These resonances disappear (due to reduction to the diamagnetic ferrous state) on enzymatic reduction with hydrogen, hydrogenase from D. vulgaris and FAD as redox mediator. The addition of nitrite to the reduced enzyme results in reoxidation of nitrite reductase as evidenced by the reappearance of most of the initial signal intensities of high-spin and low-spin ferric heme resonances. Simultaneously an intense and unusual broadened signal appears in the g=2 region, suggesting the formation of a novel heme-nitric oxide signal with the main g-value at 2.08. Confirmation of this heme-nitric oxide complex was demonstrated by reoxidation of reduced enzyme with 15 NO 2 −1 instead of 14 NO 2 −1 resulting in a decrease from three to two of the hyperfine interaction pattern of nitrogen. Thus nitrite reduction to ammonia by nitrite reductase occurs via a heme-nitric oxide intermediate as reported with spinach nitrite reductase.

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K‐12 and Wolinella succinogenes were studied by low‐temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high‐spin and low‐spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme‐NO complexes with distinct EPR spectral characteristics. The heme‐NO complexes formed with the two enzymes differed, however, in g values and line‐shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme‐NO complexes. These results suggested the involvement of an enzyme‐bound NO intermediate during the six‐electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme‐NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.

Kinetic studies of the copper nitrite reductase from Achromobacter cycloclastes and its interaction with a blue copper protein

Transient state, burst and steady state kinetics of reactions of the blue copper nitrite reductase (NIR) and blue copper protein from Achromobacter cycloclastes are investigated. The two copper-containing species are reacted with each other and where possible with dithionite, ascorbate and nitrite. Both copper proteins are fully reduced by dithionite with both S2O4(2-) and SO2-. species active. NIR is only partially reduced by ascorbate in an unusual biphasic reaction consistent with complete reduction of type-one copper followed by partial reduction of type-two copper. The rate of reduction of the type-one copper is accelerated using phenazine methosulfate as mediator. Nitrite can oxidize dithionite-reduced NIR but cannot reduce oxidized NIR. Rate constants were determined for all observed reactions.

An unusual hemoprotein capable of reversible binding of nitric oxide from the gram-positive Bacillus halodenitrificans

A green protein from the soluble extract of anaerobically grown Bacillus halodenitrificans cells was purified and determined by non-denaturing procedures or SDS-PAGE to have a molecular mass of 64 kDa. The pyridine hemochromogen was shown to be that of a b-type cytochrome prosthetic group that was soluble in ether. The protein contained 6.2mol protoheme per mol protein-1. Photoreduction of the native protein yielded a product with an electronic absorption spectrum retaining the 559 nm maximum and the 424-nm Soret band displayed in the dithionite-reduced sample. Incubation of a reduced sample in the presence of air failed to return it to the original oxidation state. Electronic spin was not affected by pH. The reduced but not the oxidized form of the cytochrome bound cyanide, carbon monoxide, and nitric oxide, providing spectra resembling those of cytochromes c′ from several sources. Addition of nitroprusside to the reduced protein yielded a spectrum similar to that of the NO reacted protein. Nitric oxide failed to reduce the green protein. The position of the Soret band in the spectrum of the nitric oxide derivative of the green protein suggested a fifth-coordinate nitrosylheme structure. EPR studies provided g values with the triplet spectral pattern consistent with a five-coordinate ferrous nitrosyl heme. Flushing of the NO-derivative with argon and overnight exposure to air returned the nitrosylheme to the ferric form, and EPR values confirmed the reversion. All these spectral characterizations are strikingly similar to those of soluble guanylate cyclase, including the observation that NO was reversibly bound to the protein. EPR spectra of whole cells also displayed the hyperfine lines typical of a nitrosyl-ferrous heme, accentuated when dithionite was added. In the absence of a definitive physiological role because of its unusual properties, the green protein was named a nitric oxide-binding protein.