Oxygen activation in neuronal NO synthase: resolving the consecutive mono-oxygenation steps.

The vital signalling molecule NO is produced by mammalian NOS (nitric oxide synthase) enzymes in two steps. L-arginine is converted into NOHA (Nω-hydroxy-L-arginine), which is converted into NO and citrulline. Both steps are thought to proceed via similar mechanisms in which the cofactor BH4 (tetrahydrobiopterin) activates dioxygen at the haem site by electron transfer. The subsequent events are poorly understood due to the lack of stable intermediates. By analogy with cytochrome P450, a haem-iron oxo species may be formed, or direct reaction between a haem-peroxy intermediate and substrate may occur. The two steps may also occur via different mechanisms. In the present paper we analyse the two reaction steps using the G586S mutant of nNOS (neuronal NOS), which introduces an additional hydrogen bond in the active site and provides an additional proton source. In the mutant enzyme, BH4 activates dioxygen as in the wild-type enzyme, but an interesting intermediate haem species is then observed. This may be a stabilized form of the active oxygenating species. The mutant is able to perform step 2 (reaction with NOHA), but not step 1 (with L-arginine) indicating that the extra hydrogen bond enables it to discriminate between the two mono-oxygenation steps. This implies that the two steps follow different chemical mechanisms.

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.

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.

Importance of the domain-domain interface to the catalytic action of the NO synthase reductase domain.

Calmodulin (CaM) activates NO synthase (NOS) by binding to a 20 amino acid interdomain hinge in the presence of Ca (2+), inducing electrons to be transferred from the FAD to the heme of the enzyme via a mobile FMN domain. The activation process is influenced by a number of structural features, including an autoinhibitory loop, the C-terminal tail of the enzyme, and a number of phosphorylation sites. Crystallographic and other recent experimental data imply that the regulatory elements lie within the interface between the FAD- and FMN-binding domains, restricting the movement of the two cofactors with respect to each other. Arg1229 of rat neuronal NOS is a conserved residue in the FAD domain that forms one of only two electrostatic contacts between the domains. Mutation of this residue to Glu reverses its charge and is expected to induce an interdomain repulsion, allowing the importance of the interface and domain-domain motion to be probed. The charge-reversal mutation R1229E has three dramatic effects on catalysis: (i) hydride transfer from NADPH to FAD is activated in the CaM-free enzyme, (ii) FAD to FMN electron transfer is inhibited in both forms, and (iii) electron transfer from FMN to the surrogate acceptor cytochrome c is activated in the CaM-free enzyme. As a result, during steady-state turnover with cytochrome c, calmodulin now deactivates the enzyme and causes cytochrome c-dependent inhibition. Evidently, domain-domain separation is large enough in the mutant to accommodate another protein between the cofactors. The effects of this single charge reversal on three distinct catalytic events illustrate how each is differentially dependent on the enzyme conformation and support a model for catalytic motion in which steps i, ii, and iii occur in the hinged open, closed, and open states, respectively. This model is also likely to apply to related enzymes such as cytochrome P450 reductase.

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.

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.

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.

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.

Probing domain mobility in a flavocytochrome.

The crystal structures of various different members of the family of fumarate reductases and succinate dehydrogenases have allowed the identification of a mobile clamp (or capping) domain [e.g., Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112], which has been proposed to be involved in regulating accessibility of the active site to substrate. To investigate this, we have constructed the A251C:S430C double mutant form of the soluble flavocytochrome c(3) fumarate reductase from Shewanella frigidimarina, to introduce an interdomain disulfide bond between the FAD-binding and clamp domains of the enzyme, thus restricting relative mobility between the two. Here, we describe the kinetic and crystallographic analysis of this double mutant enzyme. The 1.6 A resolution crystal structure of the A251C:S430C enzyme under oxidizing conditions reveals the formation of a disulfide bond, while Ellman analysis confirms its presence in the enzyme in solution. Kinetic analyses with the enzyme in both the nonbridged (free thiol) and the disulfide-bridged states indicate a slight decrease in the rate of fumarate reduction when the disulfide bridge is present, while solvent-kinetic-isotope studies indicate that in both wild-type and mutant enzymes the reaction is rate limited by proton and/or hydride transfer during catalysis. The limited effects of the inhibition of clamp domain mobility upon the catalytic reaction would indicate that such mobility is not essential for the regulation of substrate access or product release.

Oxygen activation and electron transfer in flavocytochrome P450 BM3.

In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.

Histidine 61: an important heme ligand in the soluble fumarate reductase from Shewanella frigidimarina.

An examination of the X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] shows the presence of four, bis-His-ligated, c-type hemes and one flavin adenine dinucleotide, FAD. The heme groups provide a "molecular wire" for the delivery of electrons to the FAD. Heme IV is closest to the FAD (7.4 A from heme methyl to FAD C7), and His61, a ligand to heme IV, is also close (8.4 A to FAD C7). Electron delivery to the FAD from the heme groups must proceed via heme IV, as hemes I-III are too far from the FAD for feasible electron transfer. To examine the importance of heme IV and its ligation for enzyme function, we have substituted His61 with both methionine and alanine. Here we describe the crystallographic, kinetic, and electrochemical characterization of the H61M and H61A mutant forms of the Shewanella fumarate reductase. The crystal structures of these mutant forms of the enzyme have been determined to 2.1 and 2.2 A resolution, respectively. Substitution of His61 with alanine results in heme IV having only one protein ligand (His86), the sixth coordination position being occupied by an acetate ion derived from the crystal cryoprotectant solution. In the structure of the H61M enzyme, Met61 is found not to ligate the heme iron, a role that is taken by a water molecule. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the H61M-Fcc(3) and H61A-Fcc(3) mutant enzymes are catalytically active but exhibit marked decreases in the value of k(cat) for fumarate reduction with respect to that of the wild type (5- and 10-fold lower, respectively). There is also a significant shift in the pK(a) values for the mutant enzymes, from 7.5 for the wild type to 8.26 for H61M and 9.29 for H61A. The fumarate reductase activity of both mutant enzymes can be recovered to approximately 80% of that seen for the wild type by the addition of exogenous imidazole. In the case of H61A, recovery of activity is also accompanied by a shift of the pK(a) from 9.29 to 7.46 (close, and within experimental error, to that for the wild type). Pre-steady-state kinetic measurements show clearly that rate constants for the fumarate dependent reoxidation of the heme groups are adversely affected by the mutations. The solvent isotope effect for fumarate reduction in the wild-type enzyme has a value of 8.0, indicating that proton delivery is substantially rate limiting. This value falls to 5.6 and 2.2 for the H61M and H61A mutants, respectively, indicating that electron transfer, rather than proton transfer, is becoming more rate-limiting in the mutant enzymes.

Engineering water to act as an active site acid catalyst in a soluble fumarate reductase.

The ability of an arginine residue to function as the active site acid catalyst in the fumarate reductase family of enzymes is now well-established. Recently, a dual role for the arginine during fumarate reduction has been proposed [Mowat, C. G., Moysey, R., Miles, C. S., Leys, D., Doherty, M. K., Taylor, P., Walkinshaw, M. D., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 12292-12298] in which it acts both as a Lewis acid in transition-state stabilization and as a Brønsted acid in proton delivery. This proposal has led to the prediction that, if appropriately positioned, a water molecule would be capable of functioning as the active site Brønsted acid. In this paper, we describe the construction and kinetic and crystallographic analysis of the Q363F single mutant and Q363F/R402A double mutant forms of flavocytochrome c(3), the soluble fumarate reductase from Shewanella frigidimarina. Although replacement of the active site acid, Arg402, with alanine has been shown to eliminate fumarate reductase activity, this phenomenon is partially reversed by the additional substitution of Gln363 with phenylalanine. This Gln --> Phe substitution in the inactive R402A mutant enzyme was designed to "push" a water molecule close enough to the substrate C3 atom to allow it to act as a Brønsted acid. The 2.0 A resolution crystal structure of the Q363F/R402A mutant enzyme does indeed reveal the introduction of a water molecule at the correct position in the active site to allow it to act as the catalytic proton donor. The 1.8 A resolution crystal structure of the Q363F mutant enzyme shows a water molecule similarly positioned, which can account for its measured fumarate reductase activity. However, in this mutant enzyme Michaelis complex formation is impaired due to significant and unpredicted structural changes at the active site.

Role of His505 in the soluble fumarate reductase from Shewanella frigidimarina.

The X-ray structure of the soluble fumarate reductase from Shewanella frigidimarina [Taylor, P., Pealing, S. L., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (1999) Nat. Struct. Biol. 6, 1108-1112] clearly shows the presence of an internally bound sodium ion. This sodium ion is coordinated by one solvent water molecule (Wat912) and five backbone carbonyl oxygens from Thr506, Met507, Gly508, Glu534, and Thr536 in what is best described as octahedral geometry (despite the rather long distance from the sodium ion to the backbone oxygen of Met507 (3.1 A)). The water ligand (Wat912) is, in turn, hydrogen bonded to the imidazole ring of His505. This histidine residue is adjacent to His504, a key active-site residue thought to be responsible for the observed pK(a) of the enzyme. Thus, it is possible that His505 may be important in both maintaining the sodium site and in influencing the active site. Here we describe the crystallographic and kinetic characterization of the H505A and H505Y mutant forms of the Shewanella fumarate reductase. The crystal structures of both mutant forms of the enzyme have been solved to 1.8 and 2.0 A resolution, respectively. Both show the presence of the sodium ion in the equivalent position to that found in the wild-type enzyme. The structure of the H505A mutant shows the presence of two water molecules in place of the His505 side-chain which form part of a hydrogen-bonding network with Wat48, a ligand to the sodium ion. The structure of the H505Y mutant shows the hydroxyl group of the tyrosine side-chain hydrogen-bonding to a water molecule which is also a ligand to the sodium ion. Apart from these features, there are no significant structural alterations as a result of either substitution. Both the mutant enzymes are catalytically active but show markedly different pH profiles compared to the wild-type enzyme. At high pH (above 8.5), the wild type and mutant enzymes have very similar activities. However, at low pH (6.0), the H505A mutant enzyme is some 20-fold less active than wild-type. The combined crystallographic and kinetic results suggest that His505 is not essential for sodium binding but does affect catalytic activity perhaps by influencing the pK(a) of the adjacent His504.

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.