The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase.

Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Probing the ternary complexes of indoleamine and tryptophan 2,3-dioxygenases by cryoreduction EPR and ENDOR spectroscopy.

We have applied cryoreduction/EPR/ENDOR techniques to characterize the active-site structure of the ferrous-oxy complexes of human (hIDO) and Shewanella oneidensis (sIDO) indoleamine 2,3-dioxygenases, Xanthomonas campestris (XcTDO) tryptophan 2,3-dioxygenase, and the H55S variant of XcTDO in the absence and in the presence of the substrate L-Trp and a substrate analogue, L-Me-Trp. The results reveal the presence of multiple conformations of the binary ferrous-oxy species of the IDOs. In more populated conformers, most likely a water molecule is within hydrogen-bonding distance of the bound ligand, which favors protonation of a cryogenerated ferric peroxy species at 77 K. In contrast to the binary complexes, cryoreduction of all of the studied ternary [enzyme-O(2)-Trp] dioxygenase complexes generates a ferric peroxy heme species with very similar EPR and (1)H ENDOR spectra in which protonation of the basic peroxy ligand does not occur at 77 K. Parallel studies with L-Me-Trp, in which the proton of the indole nitrogen is replaced with a methyl group, eliminate the possibility that the indole NH group of the substrate acts as a hydrogen bond donor to the bound O(2), and we suggest instead that the ammonium group of the substrate hydrogen-bonds to the dioxygen ligand. The present data show that substrate binding, primarily through this H-bond, causes the bound dioxygen to adopt a new conformation, which presumably is oriented for insertion of O(2) into the C(2)-C(3) double bond of the substrate. This substrate interaction further helps control the reactivity of the heme-bound dioxygen by "shielding" it from water.

Flavin-containing heme enzymes.

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the L-lactate dehydrogenase flavocytochrome b(2) and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.

Reassessment of the reaction mechanism in the heme dioxygenases.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme enzymes that catalyze the O(2)-dependent oxidation of L-tryptophan to N-formyl-kynurenine. Previous proposals for the mechanism of this reaction have suggested that deprotonation of the indole NH group, either by an active-site base or by oxygen bound to the heme iron, as the initial step. In this work, we have examined the activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants. We find, in contrast to previous work, that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes. These observations suggest that deprotonation of the indole N(1) is not essential for catalysis, and an alternative reaction mechanism, based on the known chemistry of indoles, is presented.

Tuning of functional heme reduction potentials in Shewanella fumarate reductases.

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.

Exploring the mechanism of tryptophan 2,3-dioxygenase.

The haem proteins TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase) are specific and powerful oxidation catalysts that insert one molecule of dioxygen into L-tryptophan in the first and rate-limiting step in the kynurenine pathway. Recent crystallographic and biochemical analyses of TDO and IDO have greatly aided our understanding of the mechanisms employed by these enzymes in the binding and activation of dioxygen and tryptophan. In the present paper, we briefly discuss the function, structure and possible catalytic mechanism of these enzymes.

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif.

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.

Histidine 55 of tryptophan 2,3-dioxygenase is not an active site base but regulates catalysis by controlling substrate binding.

Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).

Rhodobacter sphaeroides haem protein: a novel cytochrome with nitric oxide dioxygenase activity.

Rhodobacter sphaeroides produces a novel cytochrome, designated as SHP (sphaeroides haem protein), that is unusual in having asparagine as a redox-labile haem ligand. The gene encoding SHP is contained within an operon that also encodes a DHC (dihaem cytochrome c) and a membrane-associated cytochrome b. DHC and SHP have been shown to have high affinity for each other at low ionic strength (Kd=0.2 microM), and DHC is able to reduce SHP very rapidly. The reduced form of the protein, SHP2+ (reduced or ferrous SHP), has high affinity for both oxygen and nitric oxide (NO). It has been shown that the oxyferrous form, SHP2+-O2 (oxygen-bound form of SHP), reacts rapidly with NO to produce nitrate, whereas SHP2+-NO (the NO-bound form of SHP) will react with superoxide with the same product formed. It is therefore possible that SHP functions physiologically as a nitric oxide dioxygenase, protecting the organism against NO poisoning, and we propose a possible mechanism for this process.

The second enzyme in pyrrolnitrin biosynthetic pathway is related to the heme-dependent dioxygenase superfamily.

Pyrrolnitrin is a commonly used and clinically effective treatment for fungal infections and provides the structural basis for the more widely used fludioxinil. The pyrrolnitrin biosynthetic pathway consists of four chemical steps, the second of which is the rearrangement of 7-chloro-tryptophan by the enzyme PrnB, a reaction that is so far unprecedented in biochemistry. When expressed in Pseudomonas fluorescens, PrnB is red in color due to the fact that it contains 1 mol of heme b per mole of protein. The crystal structure unexpectedly establishes PrnB as a member of the heme-dependent dioxygenase superfamily with significant structural but not sequence homology to the two-domain indoleamine 2,3-dioxygenase enzyme (IDO). The heme-binding domain is also structurally similar to that of tryptophan 2,3-dioxygenase (TDO). Here we report the binary complex structures of PrnB with d- and l-tryptophan and d- and l-7-chloro-tryptophan. The structures identify a common hydrophobic pocket for the indole ring but exhibit unusual heme ligation and substrate binding when compared with that observed in the TDO crystal structures. Our solution studies support the heme ligation observed in the crystal structures. Purification of the hexahistidine-tagged PrnB yields homogeneous protein that only displays in vitro activity with 7-chloro-l-tryptophan after reactivation with crude extract from the host strain, suggesting that an as yet unknown cofactor is required for activity. Mutation of the proximal heme ligand results, not surprisingly, in inactive enzyme. Redox titrations show that PrnB displays a significantly different reduction potential to that of IDO or TDO, indicating possible differences in the PrnB catalytic cycle. This is confirmed by the absence of tryptophan dioxygenase activity in PrnB, although a stable oxyferrous adduct (which is the first intermediate in the TDO/IDO catalytic cycle) can be generated. We propose that PrnB shares a key catalytic step with TDO and IDO, generation of a tryptophan hydroperoxide intermediate, although this species suffers a different fate in PrnB, leading to the eventual formation of the product, monodechloroaminopyrrolnitrin.

An octaheme c-type cytochrome from Shewanella oneidensis can reduce nitrite and hydroxylamine.

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-A resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

Molecular mechanisms of enzyme-catalysed halogenation.

Since their discovery, halogenated metabolites have been somewhat of a biological peculiarity and it is only now that we are beginning to realize the full extent of their medicinal value. With the exception of the well characterized haloperoxidases, most of the biosynthetic enzymes and mechanisms responsible for the halogenations have remained elusive. The crystal structures of two functionally diverse halogenases have been recently solved, providing us with new and exciting mechanistic detail. This new insight has the potential to be used both in the development of biomimetic halogenation catalysts and in engineering halogenases, and related enzymes, to halogenate new substrates. Interestingly, these new structures also illustrate how the evolution of these enzymes mirrors that of the monooxygenases, where the cofactor is selected for its ability to generate a powerful oxygenating species. In this highlight article we will examine the proposed catalytic mechanisms of the halogenases and how these relate to their structures. In addition, we will consider how this chemistry might be harnessed and developed to produce novel enzymatic activity.

A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.

The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.

Structural and functional studies on DHC, the diheme cytochrome c from Rhodobacter sphaeroides, and its interaction with SHP, the sphaeroides heme protein.

The diheme cytochrome c (DHC) from Rhodobacter sphaeroides is a soluble protein with a mass of 16 kDa that represents a new class of c-type cytochrome [Vandenberghe, I., et al. (1998) Biochemistry 37, 13075-13081]. The gene encoding DHC is associated with another encoding a cytochrome known as SHP (sphaeroides heme protein). It is believed that DHC is the electron donor for SHP, which is known to bind oxygen. To gain further insight into the properties and role of DHC, we have carried out structure-function studies on the protein and examined its interaction with SHP. The crystal structures of native and recombinant DHC have been determined to resolutions of 1.85 and 2.0 A, respectively. The structures show that DHC folds into two distinct domains each containing one heme. While the N-terminal domain is a class I cytochrome c, the C-terminal domain shows no similarity to any existing structures and thus constitutes a novel cytochrome c structural motif. The shortest, edge-to-edge, distance between the heme groups is 10.2 A, and this distance is bridged by Tyr31, thus ensuring fast internal electron transfer. DHC binds strongly to its proposed physiological partner, SHP (K(d) = 0.26 microM in 10 mM HEPES at pH 7.2 and 25 degrees C). However, at higher salt concentrations, the binding becomes much weaker, indicating the importance of electrostatic interactions. DHC is also very efficient in electron transfer to SHP with a second-order rate constant of 1.8 x 10(7) M(-)(1) s(-)(1) (at pH 7.2, 10 degrees C, and I = 500 mM). The reduction potentials of DHC and SHP are also suitably ordered for a favorable reaction with the hemes of DHC showing potentials of -310 and -240 mV, respectively, and that for SHP being -105 mV. These potentials are unaltered upon complex formation.

Fumarate reductase: structural and mechanistic insights from the catalytic reduction of 2-methylfumarate.

The soluble fumarate reductase (FR) from Shewanella frigidimarina can catalyse the reduction of 2-methylfumarate with a k(cat) of 9.0 s(-1) and a K(M) of 32 microM. This produces the chiral molecule 2-methylsuccinate. Here, we present the structure of FR to a resolution of 1.5 A with 2-methylfumarate bound at the active site. The mode of binding of 2-methylfumarate allows us to predict the stereochemistry of the product as (S)-2-methylsuccinate. To test this prediction we have analysed the product stereochemistry by circular dichroism spectroscopy and confirmed the production of (S)-2-methylsuccinate.

The role of Thr268 and Phe393 in cytochrome P450 BM3.

In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (DeltaE(m) [WT+arachidonate (120 microM)]=+138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (DeltaE(m) [T268A+arachidonate (120 microM)]=+73 mV, DeltaE(m) [T268N+arachidonate (120 microM)]=+9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation.

Probing the substrate specificity of the catalytically self-sufficient cytochrome P450 RhF from a Rhodococcus sp.

Analysis of the substrate specificity of the self-sufficient cytochrome P450 RhF revealed that the enzyme tends to catalyse the dealkylation of substituted alkyl-aryl ethers with shorter alkyl moieties more readily than equivalent compounds with longer alkyl groups.

Multi-heme cytochromes--new structures, new chemistry.

Heme is one of the most pervasive cofactors in nature and the c-type cytochromes represent one of the largest families of heme-containing proteins. Recent progress in bacterial genomic analysis has revealed a vast range of genes encoding novel c-type cytochromes that contain multiple numbers of heme cofactors. The genome sequence of Geobacter sulfurreducens, for example, includes some one hundred genes encoding c-type cytochromes, with around seventy of these containing two, or more, heme groups and with one protein containing an astonishing twenty seven heme groups. This wealth of cytochromes is of great significance in the respiratory flexibility shown by bacteria such as Geobacter. In addition, we are now discovering that many of these multi-heme cytochromes have associated enzymatic activities and in some cases this is revealing new chemistries. The purpose of this perspective is to describe recent progress in the structural and functional analyses of these new multi-heme cytochromes. To illustrate this we have chosen to focus on three of these cytochromes which exhibit catalytic activities; nitrite reductase, hydroxylamine oxidoreductase and tetrathionate reductase. In addition we consider the multi-heme cytochromes from Geobacter and Desulfovibrio species. Finally, we consider and contrast the repeating structural modules found in these multi-heme cytochromes.

Analysis of the domain properties of the novel cytochrome P450 RhF.

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are -240 and -270 mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe-2S ferredoxin with a reduction potential of -214 mV.