Phenol hydroxylase from Acinetobacter radioresistens S13. Isolation and characterization of the regulatory component.

This paper reports the isolation and characterization of the regulatory moiety of the multicomponent enzyme phenol hydroxylase from Acinetobacter radioresistens S13 grown on phenol as the only carbon and energy source. The whole enzyme comprises an oxygenase moiety (PHO), a reductase moiety (PHR) and a regulatory moiety (PHI). PHR contains one FAD and one iron-sulfur cluster, whose function is electron transfer from NADH to the dinuclear iron centre of the oxygenase. PHI is required for catalysis of the conversion of phenol to catechol in vitro, but is not required for PHR activity towards alternative electron acceptors such as cytochrome c and Nitro Blue Tetrazolium. The molecular mass of PHI was determined to be 10 kDa by SDS/PAGE, 8.8 kDa by MALDI-TOF spectrometry and 18 kDa by gel-permeation. This finding suggests that the protein in its native state is a homodimer. The isoelectric point is 4.1. PHI does not contain any redox cofactor and does not bind ANS, a fluorescent probe for hydrophobic sites. The N-terminal sequence is similar to those of the regulatory proteins of phenol hydroxylase from A. calcoaceticus and Pseudomonas CF 600. In the reconstituted system, optimal reaction rate was achieved when the stoichiometry of the components was 2 PHR monomers: 1 PHI dimer: 1 PHO (alphabetagamma) dimer. PHI interacts specifically with PHR, promoting the enhancement of FAD fluorescence emission. This signal is diagnostic of a conformational change of PHR that might result in a better alignment with respect to PHO.     

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.     

Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of MMOH; and a coupling protein (MMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), -176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-)(1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.     

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.     

An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.     

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.     

Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer.     

Redox potentials of Ti(IV) and Fe(III) complexes provide insights into titanium biodistribution mechanisms

Transferrin, the human iron transport protein, binds Ti(IV) even more tightly than it binds Fe(III). However, the fate of titanium bound to transferrin is not well understood. Here we present results which address the fate of titanium once bound to transferrin. We have determined the redox potentials for a series of Ti(IV) complexes and have used these data to develop a linear free energy relationship (LFER) correlating Ti(IV) ? Ti(III) redox processes with Fe(III) ? Fe(II) redox processes. This LFER enables us to compare the redox potentials of Fe(III) complexes and Ti(IV) complexes that mimic the active site of transferrin and allows us to predict the redox potential of titanium-transferrin. Using cyclic voltammetry and discontinuous metalloprotein spectroelectrochemistry (dSEC) in conjunction with the LFER, we report that the redox potential of titanium-transferrin is lower than − 600 mV (lower than that of iron-transferrin) and is predicted to be ca. − 900 mV vs. NHE (normal hydrogen electrode). We conclude that Ti(IV)/Ti(III) reduction in titanium-transferrin is not accessible by biological reducing agents. This observation is discussed in the context of current hypotheses concerning the role of reduction in transferrin mediated iron transport.     

DNA-bound redox activity of DNA repair glycosylases containing [4Fe-4S] clusters

MutY and endonuclease III, two DNA glycosylases from Escherichia coli, and AfUDG, a uracil DNA glycosylase from Archeoglobus fulgidus, are all base excision repair enzymes that contain the [4Fe-4S](2+) cofactor. Here we demonstrate that, when bound to DNA, these repair enzymes become redox-active; binding to DNA shifts the redox potential of the [4Fe-4S](3+/2+) couple to the range characteristic of high-potential iron proteins and activates the proteins toward oxidation. Electrochemistry on DNA-modified electrodes reveals potentials for Endo III and AfUDG of 58 and 95 mV versus NHE, respectively, comparable to 90 mV for MutY bound to DNA. In the absence of DNA modification of the electrode, no redox activity can be detected, and on electrodes modified with DNA containing an abasic site, the redox signals are dramatically attenuated; these observations show that the DNA base pair stack mediates electron transfer to the protein, and the potentials determined are for the DNA-bound protein. In EPR experiments at 10 K, redox activation upon DNA binding is also evident to yield the oxidized [4Fe-4S](3+) cluster and the partially degraded [3Fe-4S](1+) cluster. EPR signals at g = 2.02 and 1.99 for MutY and g = 2.03 and 2.01 for Endo III are seen upon oxidation of these proteins by Co(phen)(3)(3+) in the presence of DNA and are characteristic of [3Fe-4S](1+) clusters, while oxidation of AfUDG bound to DNA yields EPR signals at g = 2.13, 2.04, and 2.02, indicative of both [4Fe-4S](3+) and [3Fe-4S](1+) clusters. On the basis of this DNA-dependent redox activity, we propose a model for the rapid detection of DNA lesions using DNA-mediated electron transfer among these repair enzymes; redox activation upon DNA binding and charge transfer through well-matched DNA to an alternate bound repair protein can lead to the rapid redistribution of proteins onto genome sites in the vicinity of DNA lesions. This redox activation furthermore establishes a functional role for the ubiquitous [4Fe-4S] clusters in DNA repair enzymes that involves redox chemistry and provides a means to consider DNA-mediated signaling within the cell.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

A highly sensitive nanostructure-based electrochemical sensor for electrocatalytic determination of norepinephrine in the presence of acetaminophen and tryptophan

A new electrochemical sensor for the determination of norepinephrine (NE), acetaminophen (AC) and tryptophan (Trp) is described. The sensor is based on carbon paste electrode (CPE) modified with 3,4-dihydroxybenzaldehyde-2,4-dinitrophenylhydrazone (DDP) and takes the advantages of carbon nanotubes (CNTs), which makes the modified electrode highly sensitive for the electrochemical detection of these compounds. Cyclic voltammetry (CV) at various scan rates was used to investigate the redox properties of the modified electrode. The apparent charge transfer rate constant, k(s), and transfer coefficient, α, for electron transfer between DDP and CNT paste electrode were calculated. The mediated oxidation of NE at the modified electrode was investigated by CV and the values of k, α and diffusion coefficient (D) were calculated. Under the optimum pH of 7.0, the oxidation of NE occurs at a potential about 215 mV less positive than that of the unmodified CPE. Differential pulse voltammetry (DPV) of NE at the modified electrode exhibited two linear dynamic ranges with a detection limit (3σ) of 77±2 nM. DPV was used for simultaneous determination of NE, AC and Trp at the modified electrode, and quantitation of NE in some real samples by the standard addition method.     

Antioxidant properties of carotenoids: QSAR prediction of their redox potentials

There is a great need to predict the antioxidant properties of molecules such as carotenoids. These compounds are of great interest due to their contribution to various important biological and industrial processes, including toxicity and fate. In our study, redox potentials were compiled from several literature sources. Redox potential values ranged from 537.2 mV for zeaxanthin up to 691.5 mV for beta-carotene; they correspond to the formation of cation radicals, using the standard calomel electrode (SCE). The redox potential values were measured using conventional electrochemical techniques, cyclic voltammetry and Osteryoung square-wave voltammetry. A quantitative structure-activity relationship (QSAR) was developed to model and consequently to predict the values of redox potential. The predicted values of redox potential for four external carotenoids, namely beta-carotene, zeaxanthin, cantaxanthin and astaxanthin, are presented and discussed. They indicate the dependence of redox potential on structure, donor and acceptor groups and polarisability.     

Electrochemical properties of the diiron core of uteroferrin and its anion complexes

The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and 89 mV, respectively. These shifts are consistent with the different susceptibilities of Uf to aerobic oxidation in the presence of these anions. Comparison of the reduction potential of Uf at pH 7.0 with those reported for other dinuclear non-heme iron enzymes and various (mu-oxo)diiron model complexes suggest that the potential of Uf is too positive to be consistent with a mu-oxo-bridge in Ufo. The pH dependence of the reduction potentials of Uf (60 mV/pH unit) and the fact that the electron transfer rate increases with decreasing pH indicate a concomitant participation of a proton during the oxidation-reduction process. This process was assigned to the protonation of a terminally bound hydroxide ligand at the Fe(II) center upon reduction of Ufo. Structural implications provided by the electrochemical data indicate that molybdate affects the dinuclear core in a manner that differs from that of phosphate and arsenate. This observation is consistent with previous spectroscopic and biochemical studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.     

Thermodynamic properties of the redox centers of Na(+)-translocating NADH:quinone oxidoreductase

Redox titration of all optically detectable prosthetic groups of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) at pH 7.5 showed that the functionally active enzyme possesses only three titratable flavin cofactors, one noncovalently bound FAD and two covalently bound FMN residues. All three flavins undergo different redox transitions during the function of the enzyme. The noncovalently bound FAD works as a "classical" two-electron carrier with a midpoint potential (E(m)) of -200 mV. Each of the FMN residues is capable of only one-electron reduction: one from neutral flavosemiquinone to fully reduced flavin (E(m) = 20 mV) and the other from oxidized flavin to flavosemiquinone anion (E(m) = -150 mV). The lacking second half of the redox transitions for the FMNs cannot be reached under our experimental conditions and is most likely not employed in the catalytic cycle. Besides the flavins, a [2Fe-2S] cluster was shown to function in the enzyme as a one-electron carrier with an E(m) of -270 mV. The midpoint potentials of all the redox transitions determined in the enzyme were found to be independent of Na(+) concentration. Even the components that exhibit very strong retardation in the rate of their reduction by NADH at low sodium concentrations experienced no change in the E(m) values when the concentration of the coupling ion was changed 1000 times. On the basis of these data, plausible mechanisms for the translocation of transmembrane sodium ions by Na(+)-NQR are discussed.     

Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.     

Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.     

Sequencing and analysis of the Mmethylococcus capsulatus (Bath) solublemethane monooxygenase genes.

The soluble methane monooxygenase (sMMO) hydroxylase is a prototypical member of the class of proteins with non-heme carboxylate-bridged diiron sites. The sMMO subclass of enzyme systems has several distinguishing characteristics, including the ability to catalyze hydroxylation or epoxidation chemistry, a multisubunit hydroxylase containing diiron centers in its alpha subunits, and the requirement of a coupling protein for optimal activity. Sequence homology alignment of known members of the sMMO family was performed in an effort to identify protein regions giving rise to these unique features. DNA sequencing of the Methylococcus capsulatus (Bath) sMMO genes confirmed previously identified sequencing errors and corrected two additional errors, each of which was confirmed by at least one independent method. Alignments of homologous proteins from sMMO, phenol hydroxylase, toluene 2-, 3-, and 4-monooxygenases, and alkene monooxygenase systems revealed an interesting set of absolutely conserved amino-acid residues, including previously unidentified residues located outside the diiron active site of the hydroxylase. By mapping these residues on to the M. capsulatus (Bath) sMMO hydroxylase crystal structure, functional and structural roles were proposed for the conserved regions. Analysis of the active site showed a highly conserved hydrogen-bonding network on one side of the diiron cluster but little homology on the opposite side, where substrates are presumed to bind. It is suggested that conserved residues on the hydroxylase surface may be important for protein-protein interactions with the reductase and coupling ancillary proteins and/or serve as part of an electron-transfer pathway. A possible way by which binding of the coupling protein at the surface of the hydroxylase might transfer information to the diiron active site at the interior is proposed.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.