Molecular basis for the reduced catalytic activity of the naturally occurring T560M mutant of human 12/15-lipoxygenase that has been implicated in coronary artery disease

Lipoxygenases have been implicated in cardiovascular disease. A rare single-nucleotide polymorphism causing T560M exchange has recently been described, and this mutation leads to a near null variant of the enzyme encoded for by the ALOX15 gene. When we inspected the three-dimensional structure of the rabbit ortholog, we localized Thr-560 outside the active site and identified a hydrogen bridge between its side chain and Gln-294. This interaction is part of a complex hydrogen bond network that appears to be conserved in other mammalian lipoxygenases. Gln-294 and Asn-287 are key amino acids in this network, and we hypothesized that disturbance of this hydrogen bond system causes the low activity of the T560M mutant. To test this hypothesis, we first mutated Thr-560 to amino acids not capable of forming side chain hydrogen bridges (T560M and T560A) and obtained enzyme variants with strongly reduced catalytic activity. In contrast, enzymatic activity was retained after T560S exchange. Enzyme variants with strongly reduced activity were also obtained when we mutated Gln-294 (binding partner of Thr-560) and Asn-287 (binding partner of Gln-294 and Met-418) to Leu. Basic kinetic characterization of the T560M mutant indicated that the enzyme lacks a kinetic lag phase but is rapidly inactivated. These data suggest that the low catalytic efficiency of the naturally occurring T560M mutant is caused by alterations of a hydrogen bond network interconnecting this residue with active site constituents. Disturbance of this bonding network increases the susceptibility of the enzyme for suicidal inactivation.     

Evidence for an extended hydrogen bond network in the binding site of the nicotinic receptor: role of the vicinal disulfide of the alpha1 subunit

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.     

Modeling of agonist binding to the ligand-gated ion channel superfamily of receptors

A generalized model is presented of agonist binding to ligand-gated ion channels (LGICs). Broad similarity in the structure of agonists suggests that the binding sites of LGICs may have evolved from a protobinding site. Aligned sequence data identified as a candidate for such a site a highly conserved 15 residue stretch of primary structure in the N-terminal extracellular region of all known LGIC subunits. We modeled this subregion, termed the cys-loop, as a rigid, amphiphilic beta-hairpin and propose that it may form a major determinant of a conserved structural binding cleft. In the model of the binding complex (1) an invariant aspartate residue at position 11 of the cys-loop is the anionic site interacting with the positively charged amine group of agonists, (2) a local dipole within the pi-electron system of agonists is favorably oriented in the electrostatic field of the invariant aspartate, (3) the epsilon ring-proton of a conserved aromatic residue at the turn of the cys-loop interacts orthogonally with the agonist pi-electron density at its electronegative center, and (4) selective recognition is partly a result of the type of amino acid residue at position 6 of the cys-loop. Additionally, formation of a hydrogen bond between the electronegative atom of the pi-electron system of agonist and a complementary group in the receptor may be important in the high-affinity binding of agonists.     

Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity.

The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.     

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.     

Peptide backbone chemistry and membrane channel function: effects of a single amide-to-ester replacement on gramicidin channel structure and function

To examine the structural and functional importance of backbone amide groups in ion channels for subunit folding, hydrogen bonding, ion solvation, and ion permeation, we replaced the peptide bond between Val(1) and Gly(2) in gramicidin A by an ester bond. The substitution is at the junction between the two channel subunits, where it removes an intramolecular hydrogen bond between the NH of Gly(2) and the C==O of Val(7) and perturbs an intermolecular hydrogen bond between the C==O of Val(1) in one subunit and the NH of Ala(5) in the other subunit. The substitution thus perturbs not only subunit folding but also dimer assembly, in addition to any effects on ion permeation. This backbone modification has large effects on channel function: It alters channel stability, as monitored by the channel forming ability and channel lifetime, and ion permeability, as monitored by changes in single-channel conductance and cation permeability ratios. In fact, the homodimeric channels, with two ester-containing subunits, have lifetimes so short that it becomes impossible to characterize them in any detail. The peptide --> ester substitution, however, does not affect the basic subunit fold because heterodimeric channels can form between a subunit with an ester bond and a native subunit. These heterodimeric channels, with only a single ester bond, are more easily characterized; the lone ester reduces the single-channel conductance about 4-fold and the lifetime about 200-fold as compared to the native homodimeric channels. The altered channel function results from a perturbation/disruption of the hydrogen bond network that stabilizes the backbone, as well as the membrane-spanning dimer, and that forms the lining of the ion-conducting pore. Molecular dynamics simulations show the expected destabilization of the modified heterodimeric or homodimeric channels, but the changes in backbone structure and dynamics are remarkably small. The ester bond is somewhat unstable, which precluded further structural characterization. The lability also led to a hydrolysis product that terminates with an alcohol and lacks formyl-Val. Symmetric channels formed by the hydrolyzed product again have short lifetimes, but the channels are distinctly different from those formed by the ester gramicidin A. Furthermore, well-behaved asymmetric channels form between the hydrolysis product and reference subunits that have either an L- or a D-residue at the formyl-NH-terminus.     

Refinement of a low-resolution crystal structure to better understand erythromycin interactions on large ribosomal subunit

Macrolides are a group of diverse class of naturally occurring and synthetic antibiotics made of macrocyclic-lactone ring carrying one or more sugar moieties linked to various atoms of the lactone ring. These macrolides selectively bind to a single high affinity site on the prokaryotic 50S ribosomal subunit, making them highly effective towards a wide range of bacterial pathogens. The understanding of binding between macrolides and ribosome serves a good basis in elucidating how they work at the molecular level and these findings would be important in rational drug design. Here, we report refinement of reconstructed PDB structure of erythromycin-ribosome system using molecular dynamics (MD) simulation. Interesting findings were observed in this refinement stage that could improve the understanding of the binding of erythromycin A (ERYA) onto the 50S subunit. The results showed ERYA was highly hydrated and water molecules were found to be important in bridging hydrogen bond at the binding pocket during the simulation time. ERYA binding to ribosome was also strengthened by hydrogen bond network and hydrophobic interactions between the antibiotic and the ribosome. Our MD simulation also demonstrated direct interaction of ERYA with Domains II, V and with C1773 (U1782EC), a residue in Domain IV that has yet been described of its role in ERYA binding. It is hoped that this refinement will serve as a starting model for a further enhancement of our understanding towards the binding of ERYA to ribosome.     

Structural asymmetry of phosphodiesterase-9, potential protonation of a glutamic acid, and role of the invariant glutamine

PDE9 inhibitors show potential for treatment of diseases such as diabetes. To help with discovery of PDE9 inhibitors, we performed mutagenesis, kinetic, crystallographic, and molecular dynamics analyses on the active site residues of Gln453 and its stabilizing partner Glu406. The crystal structures of the PDE9 Q453E mutant (PDE9Q453E) in complex with inhibitors IBMX and (S)-BAY73-6691 showed asymmetric binding of the inhibitors in two subunits of the PDE9Q453E dimer and also the significant positional change of the M-loop at the active site. The kinetic analysis of the Q453E and E406A mutants suggested that the invariant glutamine is critical for binding of substrates and inhibitors, but is unlikely to play a key role in the differentiation between substrates of cGMP and cAMP. The molecular dynamics simulations suggest that residue Glu406 may be protonated and may thus explain the hydrogen bond distance between two side chain oxygens of Glu453 and Glu406 in the crystal structure of the PDE9Q453E mutant. The information from these studies may be useful for design of PDE9 inhibitors.     

Computational design of second‐site suppressor mutations at protein–protein interfaces

The importance of a protein–protein interaction to a signaling pathway can be established by showing that amino acid mutations that weaken the interaction disrupt signaling, and that additional mutations that rescue the interaction recover signaling. Identifying rescue mutations, often referred to as second‐site suppressor mutations, controls against scenarios in which the initial deleterious mutation inactivates the protein or disrupts alternative protein–protein interactions. Here, we test a structure‐based protocol for identifying second‐site suppressor mutations that is based on a strategy previously described by Kortemme and Baker. The molecular modeling software Rosetta is used to scan an interface for point mutations that are predicted to weaken binding but can be rescued by mutations on the partner protein. The protocol typically identifies three types of specificity switches: knob‐in‐to‐hole redesigns, switching hydrophobic interactions to hydrogen bond interactions, and replacing polar interactions with nonpolar interactions. Computational predictions were tested with two separate protein complexes; the G‐protein Gα_i1 bound to the RGS14 GoLoco motif, and UbcH7 bound to the ubiquitin ligase E6AP. Eight designs were experimentally tested. Swapping a buried hydrophobic residue with a polar residue dramatically weakened binding affinities. In none of these cases were we able to identify compensating mutations that returned binding to wild‐type affinity, highlighting the challenges inherent in designing buried hydrogen bond networks. The strongest specificity switches were a knob‐in‐to‐hole design (20‐fold) and the replacement of a charge–charge interaction with nonpolar interactions (55‐fold). In two cases, specificity was further tuned by including mutations distant from the initial design. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A novel hypothesis for the binding mode of HERG channel blockers

We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a pi-pi interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-pi interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC(50) value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC(50), and the group having more predicted interactions with the HERG channel had a higher mean pIC(50) value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers.     

Solution NMR structure and X-ray absorption analysis of the C-terminal zinc-binding domain of the SecA ATPase

The solution NMR structure of a 22-residue Zn(2+)-binding domain (ZBD) from Esherichia coli preprotein translocase subunit SecA is presented. In conjunction with X-ray absorption analysis, the NMR structure shows that three cysteines and a histidine in the sequence CXCXSGX(8)CH assume a tetrahedral arrangement around the Zn(2+) atom, with an average Zn(2+)-S bond distance of 2.30 A and a Zn(2+)-N bond distance of 2.03 A. The NMR structure shows that ND1 of His20 binds to the Zn(2+) atom. The ND1-Zn(2+) bond is somewhat strained: it makes an angle of approximately 17 degrees with the plane of the ring, and it also shows a significant "in-plane" distortion of 13 degrees. A comprehensive sequence alignment of the SecA-ZBD from many different organisms shows that, along with the four Zn(2+) ligands, there is a serine residue (Ser12) that is completely conserved. The NMR structure indicates that the side chain of this serine residue forms a strong hydrogen bond with the thiolate of the third cysteine residue (Cys19); therefore, the conserved serine appears to have a critical role in the structure. SecB, an export-specific chaperone, is the only known binding partner for the SecA-ZBD. A phylogenetic analysis using 86 microbial genomes shows that 59 of the organisms carry SecA with a ZBD, but only 31 of these organisms also possess a gene for SecB, indicating that there may be uncharacterized binding partners for the SecA-ZBD.     

The hydrogen bonds between Arg423 and Glu472 and other key residues, Asp443, Ser477, and Pro489, are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na(+)/K(+)-ATPase

Mutation of Arg(423) at the N-domain of Na(+)/K(+)-ATPase resulted in a large decrease of both TNP-ATP and ATP binding. Thus, this residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site. In addition, mutation of Glu(472) also caused a large decrease of both TNP-ATP and ATP binding. On the basis of our computer model, we hypothesized that a hydrogen bond between Arg(423) and Glu(472) supports the connection of two opposite halves of the ATP-binding pocket. To verify this hypothesis, we have also prepared the construct containing both these mutations. Binding of neither TNP-ATP nor ATP to this double mutant differed from binding to any of the single mutants. This strongly supported the existence of the hydrogen bond between Arg(423) and Glu(472). Similarly, the conserved residue Pro(489) seems to be substantial for the proper interaction of the third and fourth beta-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp(443) affected only ATP, but not TNP-ATP, binding, suggesting that these ligands adopt different positions in the nucleotide-binding pocket. On the basis of a recently published crystal structure [H?kansson, K. O. (2003) J. Mol. Biol. 332, 1175-1182], we improved our model and computed the interaction of these two ligands with the N-domain. This model is in good agreement with all previously reported spectroscopic data and revealed that Asp(443) forms a hydrogen bond with the NH(2) group of the adenosine moiety of ATP, but not TNP-ATP.     

Contributions of the interdomain loop, amino terminus, and subunit interface to the ligand-facilitated dimerization of neurophysin: crystal structures and mutation studies of bovine neurophysin-I

Current evidence indicates that the ligand-facilitated dimerization of neurophysin is mediated in part by dimerization-induced changes at the hormone binding site of the unliganded state that increase ligand affinity. To elucidate other contributory factors, we investigated the potential role of neurophysin's short interdomain loop (residues 55-59), particularly the effects of loop residue mutation and of deleting amino-terminal residues 1-6, which interact with the loop and adjacent residues 53-54. The neurophysin studied was bovine neurophysin-I, necessitating determination of the crystal structures of des 1-6 bovine neurophysin-I in unliganded and liganded dimeric states, as well as the structure of its liganded Q58V mutant, in which peptide was bound with unexpectedly increased affinity. Increases in dimerization constant associated with selected loop residue mutations and with deletion of residues 1-6, together with structural data, provided evidence that dimerization of unliganded neurophysin-I is constrained by hydrogen bonding of the side chains of Gln58, Ser56, and Gln55 and by amino terminus interactions, loss or alteration of these hydrogen bonds, and probable loss of amino terminus interactions, contributing to the increased dimerization of the liganded state. An additional intersubunit hydrogen bond from residue 81, present only in the liganded state, was demonstrated as the largest single effect of ligand binding directly on the subunit interface. Comparison of bovine neurophysins I and II indicates broadly similar mechanisms for both, with the exception in neurophysin II of the absence of Gln55 side chain hydrogen bonds in the unliganded state and a more firmly established loss of amino terminus interactions in the liganded state. Evidence is presented that loop status modulates dimerization via long-range effects on neurophysin conformation involving neighboring Phe22 as a key intermediary.     

Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 A resolution

The binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X-ray crystallography. The three-dimensional structure of the complex has been refined at 1.9 A resolution to a crystallographic R-factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co-ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side-chain of Arg66 (E10) which, from an outer orientation in the 'aquo-met' ligand-free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.     

Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.     

Stereospecific interactions of proline residues in protein structures and complexes

The constrained backbone torsion angle of a proline (Pro) residue has usually been invoked to explain its three-dimensional context in proteins. Here we show that specific interactions involving the pyrrolidine ring atoms also contribute to its location in a given secondary structure and its binding to another molecule. It is adept at participating in two rather non-conventional interactions, C-H...pi and C-H...O. The geometry of interaction between the pyrrolidine and aromatic rings, vis-à-vis the occurrence of the C-H...pi interactions has been elucidated. Some of the secondary structural elements stabilized by Pro-aromatic interactions are beta-turns, where a Pro can interact with an adjacent aromatic residue, and in antiparallel beta-sheet, where a Pro in an edge strand can interact with an aromatic residue in the adjacent strand at a non-hydrogen-bonded site. The C-H groups at the Calpha and Cdelta positions can form strong C-H...O interactions (as seen from the clustering of points) and such interactions involving a Pro residue at C' position relative to an alpha-helix can cap the hydrogen bond forming potentials of the free carbonyl groups at the helix C terminus. Functionally important Pro residues occurring at the binding site of a protein almost invariably engage aromatic residues (with one of them being held by C-H...pi interaction) from the partner molecule in the complex, and such aromatic residues are highly conserved during evolution.     

A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.     

Molecular structure of flavocytochrome b2 at 2.4 A resolution

The crystal structure of flavocytochrome b2 has been solved at 3.0 A resolution by the method of multiple isomorphous replacement with anomalous scattering. Area detector data from native and two heavy-atom derivative crystals were used. The phases were refined by the B.C. Wang phase-filtering procedure utilizing the 67% (v/v) solvent content of the crystals. A molecular model was built first on a minimap and then on computer graphics from a combination of maps both averaged and not averaged about the molecular symmetry axis. The structure was extended to 2.4 A resolution using film data recorded at a synchrotron and refined by the Hendrickson-Konnert procedure. The molecule, a tetramer of Mr 230,000, is located on a crystallographic 2-fold axis and possesses local 4-fold symmetry. Each subunit is composed of two domains, one binding a heme and the other an FMN prosthetic group. In subunit 1, both the cystochrome and the flavin-binding domain are visible in the electron density map. In subunit 2 the cytochrome domain is disordered. However, in the latter, a molecule of pyruvate, the product of the enzymatic reaction, is bound at the active site. The cytochrome domain consists of residues 1 to 99 and is folded in a fashion similar to the homologous soluble fragment of cytochrome b5. The flavin binding domain contains a parallel beta 8 alpha 8 barrel structure and is composed of residues 100 to 486. The remaining 25 residues form a tail that wraps around the molecular 4-fold axis and is in contact with each remaining subunit. The FMN moiety, which is located at the C-terminal end of the central beta-barrel, is mostly sequestered from solvent; it forms hydrogen bond interactions with main- and side-chain atoms from six of the eight beta-strands. The interaction of Lys349 with atoms N-1 and O-2 of the flavin ring is probably responsible for stabilization of the anionic form of the flavin semiquinone and hydroquinone and enhancing the reactivity of atom N-5 toward sulfite. The binding of pyruvate at the active site in subunit 2 is stabilized by interaction of its carboxylate group with the side-chain atoms of Arg376 and Tyr143. Residues His373 and Tyr254 interact with the keto-oxygen atom and are involved in catalysis. In contrast, four water molecules occupy the substrate-binding site in subunit 1 and Tyr143 forms a hydrogen bond to the ordered heme propionate group. Otherwise the two flavin-binding domains are identical within experimental error.(ABSTRACT TRUNCATED AT 400 WORDS)