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.

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.

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.

Octaheme tetrathionate reductase is a respiratory enzyme with novel heme ligation.

We have isolated a soluble cytochrome from Shewanella oneidensis that contains eight covalently attached heme groups and determined its crystal structure. One of these hemes exhibits novel ligation of the iron atom by the epsilon-amino group of a lysine residue, despite its attachment via a typical CXXCH motif. This heme is most likely the active site for tetrathionate reduction, a reaction catalyzed efficiently by this enzyme.

P450 BM3: the very model of a modern flavocytochrome.

Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH-P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.

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).

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.

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.

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.

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.

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.

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.

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.

Determination of the orientation of the axial ligands and of the magnetic properties of the haems in the tetrahaem ferricytochrome from Shewanella frigidimarina.

The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the alpha-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic (13)C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins.

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR.

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.

Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI.

The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity. N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence. Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster. The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a [4Fe-4S](+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88. Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a [4Fe-4S](+) cluster. Potentiometric titrations indicate that Fer has a reduction potential of -385+/-10 mV for the [4Fe-4S](+)-[4Fe-4S](2+) redox couple, well within the normal range expected for such a ferredoxin. A proposed physiological role for Fer is as an electron donor to cytochrome P450 BioI. Studies on Fer binding to P450 BioI give rise to a K(d) value of 0.87+/-0.10 microM. Anaerobic experiments using CO-saturated buffer indicate that Fer is indeed capable of transferring electrons to this cytochrome P450 albeit at a fairly low rate.

Catalytically functional flavocytochrome chimeras of P450 BM3 and nitric oxide synthase.

P450 BM3 and the nitric oxide synthases are related classes of flavocytochrome mono-oxygenase enzymes, containing NADPH-dependent FAD- and FMN-containing oxidoreductase modules fused to heme b-containing oxygenase domains. Domain-swap hybrids of these two multi-domain enzymes were created by genetic engineering of different segments of reductase and heme domains from neuronal nitric oxide synthase and P450 BM3, as a means of investigating the catalytic competence and substrate-binding properties of the fusions and the influence of tetrahydrpbiopterin and calmodulin binding regions on the electron transfer kinetics of the chimeras. Despite marked differences in hybrid stability and solubility, four catalytically functional chimeras were created that retained good reductase activity and which could be expressed successfully in Escherichia coli and purified. All of the BM3 reductase domain chimeras (chimeras I-III) exhibited inefficient flavin-to-heme inter-domain electron transfer, diminishing their oxygenase activity. However, the chimera containing the neuronal nitric oxide synthase reductase domain (chimera IV) showed good oxygenase domain activity, indicating that the flavin-to-heme electron transfer reaction is relatively efficient in this case. The data reinforce the importance of the nature of inter-domain linker constitution in multi-domain enzymes, and the difficulties posed in attempts to create chimeric enzymes with enhanced catalytic properties.