Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme.

A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible one-proton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site.

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-Ser(719), NarG-His(1163), and NarG-His(1184)); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His(1092) and NarG-His(1098)). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔEm values of -88 and -36 mV, respectively). Ala variants of His(1092) and His(1098) also elicit large ΔEm values of -143 and -101 mV, respectively. An Arg variant of His(1092) elicits a small ΔEm of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum Em 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.

Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase.

The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (-87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The "free" and "occupied" states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD.

Shifting the metallocentric molybdoenzyme paradigm: the importance of pyranopterin coordination.

In this review, we test the hypothesis that pyranopterin coordination plays a critical role in defining substrate reactivities in the four families of mononuclear molybdenum and tungsten enzymes (Mo/W-enzymes). Enzyme families containing a single pyranopterin dithiolene chelate have been demonstrated to have reactivity towards two (sulfite oxidase, SUOX-fold) and five (xanthine dehydrogenase, XDH-fold) types of substrate, whereas the major family of enzymes containing a bis-pyranopterin dithiolene chelate (dimethylsulfoxide reductase, DMSOR-fold) is reactive towards eight types of substrate. A second bis-pyranopterin enzyme (aldehyde oxidoreductase, AOR-fold) family catalyzes a single type of reaction. The diversity of reactions catalyzed by each family correlates with active site variability, and also with the number of pyranopterins and their coordination by the protein. In the case of the AOR-fold enzymes, inflexibility of pyranopterin coordination correlates with their limited substrate specificity (oxidation of aldehydes). In examples of the SUOX-fold and DMSOR-fold enzymes, we observe three types of histidine-containing charge-transfer relays that can: (1) connect the piperazine ring of the pyranopterin to the substrate-binding site (SUOX-fold enzymes); (2) provide inter-pyranopterin communication (DMSOR-fold enzymes); and (3) connect a pyran ring oxygen to deeply buried water molecules (the DMSOR-fold NarGHI-type nitrate reductases). Finally, sequence data mining reveals a number of bacterial species whose predicted proteomes contain large numbers (up to 64) of Mo/W-enzymes, with the DMSOR-fold enzymes being dominant. These analyses also reveal an inverse correlation between Mo/W-enzyme content and pathogenicity.

Pyranopterin conformation defines the function of molybdenum and tungsten enzymes.

We have analyzed the conformations of 319 pyranopterins in 102 protein structures of mononuclear molybdenum and tungsten enzymes. These span a continuum between geometries anticipated for quinonoid dihydro, tetrahydro, and dihydro oxidation states. We demonstrate that pyranopterin conformation is correlated with the protein folds defining the three major mononuclear molybdenum and tungsten enzyme families, and that binding-site micro-tuning controls pyranopterin oxidation state. Enzymes belonging to the bacterial dimethyl sulfoxide reductase (DMSOR) family contain a metal-bis-pyranopterin cofactor, the two pyranopterins of which have distinct conformations, with one similar to the predicted tetrahydro form, and the other similar to the predicted dihydro form. Enzymes containing a single pyranopterin belong to either the xanthine dehydrogenase (XDH) or sulfite oxidase (SUOX) families, and these have pyranopterin conformations similar to those predicted for tetrahydro and dihydro forms, respectively. This work provides keen insight into the roles of pyranopterin conformation and oxidation state in catalysis, redox potential modulation of the metal site, and catalytic function.

A new paradigm for electron transfer through Escherichia coli nitrate reductase A.

We have investigated the role of redox cooperativity in defining the functional relationship among the three membrane-associated prosthetic groups of Escherichia coli nitrate reductase A: the two hemes (bD and bP) of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster (FS4) of the electron-transfer subunit (NarH). Previously published analyses of potentiometric titrations have exhibited the following anomalous behaviors: (i) fits of titration data for heme bp and the [3Fe-4S] cluster exhibited two apparent components; (ii) heme bD titrated with an apparent electron stoichiometry (n) of <1.0; and (iii) the binding of quinol oxidation inhibitors shifted the reduction potentials of both hemes despite there being only a single quinol oxidation site (Q-site) in close juxtaposition with heme bD. Furthermore, both hemes appeared to be affected despite the absence of major structural shifts upon inhibitor binding, as judged by X-ray crystallography, or evidence of a second Q-site in the vicinity of heme bP. In a re-examination of the redox behavior of hemes bD and bP and FS4, we have developed a cooperative redox model of cofactor interaction. We show that anticooperative interactions provide an explanation for the anomalous behavior. We propose that the role of such anticooperative redox behavior in vivo is to facilitate transmembrane electron transfer across an energy-conserving membrane against an electrochemical potential.

Q-site occupancy defines heme heterogeneity in Escherichia coli nitrate reductase A (NarGHI).

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.

Electron-transfer pathways in the heme and quinone-binding domain of complex II (succinate dehydrogenase).

Single electron transfers have been examined in complex II (succinate:ubiquinone oxidoreductase) by the method of pulse radiolysis. Electrons are introduced into the enzyme initially at the [3Fe-4S] and ubiquinone sites followed by intramolecular equilibration with the b heme of the enzyme. To define thermodynamic and other controlling parameters for the pathways of electron transfer in complex II, site-directed variants were constructed and analyzed. Variants at SdhB-His207 and SdhB-Ile209 exhibit significantly perturbed electron transfer between the [3Fe-4S] cluster and ubiquinone. Analysis of the data using Marcus theory shows that the electronic coupling constants for wild-type and variant enzyme are all small, indicating that electron transfer occurs by diabatic tunneling. The presence of the ubiquinone is necessary for efficient electron transfer to the heme, which only slowly equilibrates with the [3Fe-4S] cluster in the absence of the quinone.

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.

A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase.

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe-4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe-4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe-4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe-4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (>100mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.

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.

Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster.

Succinate-ubiquinone oxidoreductase (SQR) and menaquinol-fumarate oxidoreductase (QFR) from Escherichia coli are members of the complex II family of enzymes. SQR and QFR catalyze similar reactions with quinones; however, SQR preferentially reacts with higher potential ubiquinones, and QFR preferentially reacts with lower potential naphthoquinones. Both enzymes have a single functional quinone-binding site proximal to a [3Fe-4S] iron-sulfur cluster. A difference between SQR and QFR is that the redox potential of the [3Fe-4S] cluster in SQR is 140 mV higher than that found in QFR. This may reflect the character of the different quinones with which the two enzymes preferentially react. To investigate how the environment around the [3Fe-4S] cluster affects its redox properties and catalysis with quinones, a conserved amino acid proximal to the cluster was mutated in both enzymes. It was found that substitution of SdhB His-207 by threonine (as found in QFR) resulted in a 70-mV lowering of the redox potential of the cluster as measured by EPR. The converse substitution in QFR raised the redox potential of the cluster. X-ray structural analysis suggests that placing a charged residue near the [3Fe-4S] cluster is a primary reason for the alteration in redox potential with the hydrogen bonding environment having a lesser effect. Steady state enzyme kinetic characterization of the mutant enzymes shows that the redox properties of the [3Fe-4S] cluster have only a minor effect on catalysis.

Protein crystallography reveals a role for the FS0 cluster of Escherichia coli nitrate reductase A (NarGHI) in enzyme maturation.

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His(49) both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E(m) value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E(m) results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg(94)) in the vicinity of FS0 to a Ser residue. In this case, the E(m) of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to approximately 30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, (49)HGVNCTG(55), must be correctly positioned to ensure holoenzyme maturation.

Escherichia coli succinate dehydrogenase variant lacking the heme b.

The Escherichia coli enzyme succinate:ubiquinone oxidoreductase [(succinate dehydrogenase (SdhCDAB)] couples succinate oxidation to ubiquinone reduction and is structurally and functionally equivalent to mitochondrial complex II, an essential component of the aerobic respiratory chain and tricarboxylic acid cycle. All such enzymes contain a heme within their membrane anchor domain with a highly contentious, but as-yet-undetermined, function. Here, we report the generation of a complex II that lacks heme, which is confirmed by both optical and EPR spectroscopy. Despite the absence of heme, this mutant still assembles properly and retains physiological activity. However, the mutants lacking heme are highly sensitive to the presence of detergent. In addition, the heme does not appear to be involved in reactive oxygen species suppression. Our results indicate that redox cycling of the heme in complex II is not essential for the enzyme's ubiquinol reductase activity.

The prokaryotic complex iron-sulfur molybdoenzyme family.

Bacterial genomes encode an extensive range of respiratory enzymes that enable respiratory metabolism with a diverse group of reducing and oxidizing substrates under both aerobic and anaerobic growth conditions. An important class of enzymes that contributes to this broad diversity is the complex iron-sulfur molybdoenzyme (CISM) family. The architecture of this class comprises the following subunits. (i) A molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor-containing catalytic subunit that also contains a cubane [Fe-S] cluster (FS0). (ii) A four-cluster protein (FCP) subunit that contains 4 cubane [Fe-S] clusters (FS1-FS4). (iii) A membrane anchor protein (MAP) subunit which anchors the catalytic and FCP subunits to the cytoplasmic membrane. In this review, we define the CISM family of enzymes on the basis of emerging structural and bioinformatic data, and show that the catalytic and FCP subunit architectures appear in a wide range of bacterial redox enzymes. We evaluate evolutionary events involving genes encoding the CISM catalytic subunit that resulted in the emergence of the complex I (NADH:ubiquinone oxidoreductase) Nqo3/NuoG subunit architecture. We also trace a series of evolutionary events leading from a primordial Cys-containing peptide to the FCP architecture. Finally, many of the CISM archetypes and related enzymes rely on the tat translocon to transport fully folded monomeric or dimeric subunits across the cytoplasmic membrane. We have used genome sequence data to establish that there is a bias against the presence of soluble periplasmic molybdoenzymes in bacteria lacking an outer membrane.

Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase.

Escherichia coli succinate dehydrogenase (Sdh) belongs to the highly conserved complex II family of enzymes that reduce ubiquinone. These enzymes do not generate a protonmotive force during catalysis and are electroneutral. Because of its electroneutrality, the quinone reduction reaction must consume cytoplasmic protons which are released stoichiometrically during succinate oxidation. The X-ray crystal structure of E. coli Sdh shows that residues SdhB (G227), SdhC (D95), and SdhC (E101) are located at or near the entrance of a water channel that has been proposed to function as a proton wire connecting the cytoplasm to the quinone binding site. However, the pig and chicken Sdh enzymes show an alternative entrance to the water channel via the conserved SdhD (Q78) residue. In this study, site-directed mutants of these four residues were created and characterized by in vivo growth assays, in vitro activity assays, and electron paramagnetic resonance spectroscopy. We show that the observed water channel in the E. coli Sdh structure is the functional proton wire in vivo, while in vitro results indicate an alternative entrance for protons. In silico examination of the E. coli Sdh reveals a possible H-bonding network leading from the cytoplasm to the quinone binding site that involves SdhD (D15). On the basis of these results we propose an alternative proton pathway in E. coli Sdh that might be functional only in vitro.

Detection and interpretation of redox potential optima in the catalytic activity of enzymes.

It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.

Defining the Q-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy.

We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K(d) of 2.5 nM, and the following mutations significantly increase this value: FrdB-T205H (K(d) = 39 nM); FrdB-V207C (K(d) = 20 nM); FrdC-E29L (K(d) = 25 nM); FrdC-W86R (no detectable binding); and FrdD-H80K (K(d) = 20 nM). In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K(m) and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.

Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase.

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.

Evolutionary persistence of the molybdopyranopterin-containing sulfite oxidase protein fold.

SUMMARY: The importance of molybdoenzymes is exemplified both by the debilitating and fatal human diseases caused by their deficiency and by their persistence throughout evolution. Here, we show that the protein fold of the molybdopyranopterin-containing domain of sulfite oxidase (the SUOX fold) can be found in all three domains of life. Analyses of sequence data and protein structure comparisons (secondary structure matching) show that the SUOX fold is found in enzymes that have quite distinct macromolecular architectures comprising one or more domains and sometimes subsidiary subunits. These are summarized as follows: (i) animal SUOXs that contain an N-terminal cytochrome b(5) domain and an SUOX fold fused to a C-terminal dimerization domain; (ii) plant SUOX that contains an SUOX fold fused to a C-terminal dimerization domain; (iii) the YedY protein from Escherichia coli, which comprises only the SUOX fold; (iv) the sulfite dehydrogenase from Starkeya novella that contains the SUOX fold, a dimerization domain, and an additional c-type cytochrome subunit; and (v) the plant-type nitrate reductases, exemplified by that of Pichia angusta, that contain an N-terminal SUOX fold, a dimerization domain, a cytochrome b(5) domain, and a C-terminal NADH binding flavin adenine dinucleotide-containing domain. We used the primary sequences of the proteins containing an SUOX fold to mine 559 sequences of related proteins. A phylogeny of a nonredundant subset of these sequences was generated, and the resultant clades were categorized by sequence motif analyses in the context of the available protein structures. Based on the motif analyses, cladistics, and domain conservations, we are able to postulate a plausible pathway of SUOX fold enzyme evolution.