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Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-l; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone. doi: 10.1128/JB.00589-10

By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the major pathway for cholesterol removal from the brain. Two crystal structures of CYP46A1 were determined. First is the 1.9-[Angstrom] structure of CYP46A1 complexed with a high-affinity substrate cholesterol 3-sulfate (CH-3S). The second structure is that of the substrate-free CYP46A1 at 2.4-[Angstrom] resolution. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A unique helix B'-C loop insertion (residues 116-120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. A comparison with the substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. In vitro assays were performed to characterize the effect of different therapeutic agents on cholesterol hydroxylase activity of purified full-length recombinant CYP46A1, and several strong inhibitors and modest coactivators of CYP46A1 were identified. Structural and biochemical data provide evidence that CYP46A1 activity could be altered by exposure to some therapeutic drugs and potentially other xenobiotics. cholesterol metabolism | monooxygenase | drug interactions | cholesterol 3-sulfate

A study investigates whether the near-IR fluorescent probe Nile Red is an allosteric substrate of cytochrome P450 3A4 (CYP3A4). It is concluded that Nile Red, which undergoes sequential oxidation and exhibits complex binding behavior, provides the advantage of extreme solvatochromism and can be used to explore the properties any sites on CYP3A4 to which it binds.

Studies of 25-hydroxyvitamin [D.sub.3]-24-hydroxylase (CYP24A1) have demonstrated that it is a bifunctional enzyme capable of the 24-hydroxylation of 1[alpha],25-[(OH).sub.2][D.sub.3], leading to the excretory form, calcitroic acid, and 23-hydroxylation, culminating in 1[alpha],25-[(OH).sub.2][D.sub.3]-26,23-lactone. The degree to which CYP24A1 performs either 23- or 24-hydroxylation is species-dependent. In this paper, we show that the human enzyme that predominantly 24-hydroxylates its substrate differs from the opossum enzyme that 23-hydroxylates it at only a limited number of amino acid residues. Mutagenesis of the human form at a single substrate-binding residue (A326G) dramatically changes the regioselectivity of the enzyme from a 24-hydroxylase to a 23-hydroxylase, whereas other modifications have no effect. Ala-326 is located in the I-helix, close to the terminus of the docked 25-hydroxylated side chain in a CYP24A1 homology model, a result that we interpret indicates that substitution of a glycine at 326 provides extra space for the side chain of the substrate to move deeper into the pocket and place it in a optimal stereochemical position for 23-hydroxylation. We discuss the physiological ramifications of these results for species possessing the A326G substitution, as well as implications for optimal vitamin D analog design. 1[alpha],25-[(OH).sub.2][D.sub.3] | cytochrome P450 | dual metabolic pathways | substrate docking

The tetraheme cytochrome c(sub 554) [cyt c(sub 554)] from Nitrosomonas europae is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase. The NO reductase activity of cyt c(sub 554) may be important during ammonia oxidation in N and europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine.

Two well-studied small alpha-helical proteins, the B-domain of protein A and Rd-apocytochrome b562 were coupled by fusing the C-terminal helix of the B-domain of protein A with the N-terminal helix of Rd-apocyt b562 without changing their hydrophobic core residues. The results suggest that the two domains have weakenergetic coupling.

The pH dependencies of redox difference spectra is analyzed to study the redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc1 complex by using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Histidine is relatively sharp and has a high extinction coefficient and also, provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms.

X-ray structures at 3.0-3.1 angstrom resolution of the cytochrome b6f complex from the cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii showed the presence of a unique heme, heme x. The only axial ligand associated with this heme is a H2O or OH(super -) that is H-bonded to the propionate of the stormal side heme bn, showing that it is pentacoordinate.

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(sub d) of 16.4 MuM at 25 degree C, pH 6.0, in an endothermic rdcytochromeeaction that is driven by a large entropy change. Cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required restoring the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

A detailed structural, spectroscopic and thermodynamic characterization of cytochrome P450 BioI is conducted and reported. Results demonstrate unexpected flexibility of the active site with the capacity to bind tightly to certain steroids and to bulky azole drugs, as well as long-chain fatty acids. The result also reveals that BioI is purified from Escherichia coli with palmitic acid bound, which explains its isolation in a mixed spin state.

The use of mathematical parametrization of the backbone coordinates of the transmembrane diheme four-helix bundle in cytochrome bc(sub 1) as a starting point for the design of a water-soluble model for this protein is presented. NMR spectra reveal that the protein exists in solution in a unique conformation and is guided to structural studies.

Regioselective and enantioselective alkane hydroxylation in the presence of atmospheric dioxygen as an oxidant is performed by using cytochrome P450 MB-3 from Bacillus megaterium, which is engineered by applying a combination of directed evolution and site-directed mutagenesis. It can be concluded that the functional flexibility of the BM-3 scaffold is exhibited by the regioselective and enantioselective alkane hydroxylation activities.

The (NMR) is used to investigate the complex of the photosynthetic redox partners plastocyanin and cytochrome f (cytf) from the thermophilic cyanobacterium, Phormidium laminosum. It was found that the complex interface involves the hydrophobic patch of poplar (Pc) and the heme region of cytf and also the plastocyanin-cytf complex from P. laminosum was predominantly controlled by hydrophobic interactions.

The time-resolved reaction between fully reduced cytochrome [ba.sub.3] from Thermus thermophilus and [O.sub.2] in variants where Thr-312 and Thr-315 are modified is examined. The results have shown that proton uptake is inhibited in the two variants, which has suggested involvement of T312 and T315 in the transfer of protons to the achieve site during [O.sub.2] reduction in the [ba.sub.3] oxidase.

NMR spectroscopy is used to determine the the p[K.sub.a] values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtRp). The likely translocation of TtRp between the cytochrome b and cytochrome c sites is most consistent with a mechanism involving the coupled transfer of an electron and transfer of the proton across the hydrogen bond between the hydroquinone and His154 at the cytochrome b site.

A strategy is described by which reactive binding of a weakly bound, 'dynamically docked (DD)', complex without a known structure is strengthened electrostatically through optimized placement of surface charges, and its utility in modulating complex formation between myoglobin (Mb) and cytochrome [b.sub.5] ([b.sub.5]) is described. The measurements have shown that the presence of fast photoinduced singlet-electron transfer (ET) across the protein-protein interface.

Two-dimensional proton-evolved local-field (2D PELF) pulse sequences are implemented by using either composite zero cross-polarization (COMPOZER-CP) or windowless isotropic mixing (WIM) for magnetization transfer. The results from magnetically aligned bicelles containing uniformly [super 15]N-labeled cytochrome [b.sub.5] have shown that the PELF-based experimental approaches have a major impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically aligned bicelles.

The system I cytochrome c maturation (Ccm) apparatus has handled a wide variety of apocytochrome substrates containing the C[X.sub.n]CH heme attachment sequence. The unprecedented, aberrant persulfide linkages have helped in understanding the mechanism of the attachment of heme to substrate apocytochrome within the Ccm complex of Escherichia coli.

A study was conducted to understand the kinetic mechanism related to the role of structurally homologous flavoproteins photolyases and cryptochromes (CRY) towards UV-damaged DNA repair by photoinduced electron transfer from their FAD cofactor. The results obtained offer insight into the kinetic tuning of the rates of electron transfer which would play a function-defining role in photolyases and cryptochromes.

A detailed model of electron transfer (ET) events that was applied to all of the generated protein configurations was used to compute ET rates and understand Brownian dynamics simulations of the diffusional association of plastocyanin (PC) and cytochrome f (CytF) from the cyanobacterium Phormidium laminosum. The simulations offer insight into the strong electrostatic interactions between P. laminosum PC and CytF which influenced the ET rates from both diffusion and thermal activation processes.

The development of a convenient cell-based assay cultured with adhesive cells, HepG2, in the conventional culture dish containing glass disks is presented. The assay helped to measure the cytochrome P4501A1 (CYP1A1)-mediated activity of ethoxyresorufin O-deethylation (EROD), in HepG2.

Cryogenic Fourier transform infrared (FTIR) photolysis experiments on fully reduced [bo.sub.3]-NO and the mixed gas [bo.sub.3]-CO/NO complexes are described. The difference in metal-metal distance has not affected the NO reductase activity and hence the results have shown that the coordination of the second NO to [Cu.sub.B] is not a necessary step of the reaction mechanism.

Microspectrophotometry of single crystals of cytochrome [ba.sub.3] oxidase from Thermus thermophilus has shown that crystals of the oxidized enzyme are reduced in an intense X-ray. An unexpected optical absorption envelope at approximately 590 nm is observed from a five-coordinate, low-spin, ferrous heme [a.sub.3] that is trapped in the frozen crystals.

The solution structure of the reduced form of Shewanella frigidimarina NCIMB400 (Sfc) is analyzed. The studies have shown that the small tetraheme cytochromes isolated from Shewanella spp. has represented a new structural motif that is distinct from that of the tetraheme cytochromes [c.sub.3] from the Desulfovibrionacea family.

A stopped-flow technique in conjunction with theoretical computation method is used to examine the kinetics of reduction of a series of Fe(III) complexes with citrate, nitrilotriacetic acid (NTA), and EDTA by MtrC and OmcA. The results revealed that ligand complexation and relatively large reorganization energy contributes significantly to microbial dissimilatory reduction and mineral transformation of iron, as well as other redox-sensitive metal species in nature.

The glutamic acid residue of transmembrane helix III in subunit I (E107L) mutant cytochrome bd from Escherichia coli is studied by using electrometry and absorption spectroscopy with a microsecond time resolution to elucidate the role of E107 in translocation of protons within the protein molecule. The studies have shown that E107 is either the second group or a key residue of a proton delivery pathway leading from the cytoplasm toward the second group.

The spectroelectrochemical studies of Escherichia coli NrfA have provided unprecedented resolution of its thermodynamic properties. These methodologies have also facilitated studies of other oxidoreductases for which catalytic protein film voltammetry (PFV) is defined.

The calculation result for P450 StaP (CYP254A1), the family of cytochromes [P450.sup.1a] that catalyzes the formation of staurosporine from chromopyrrolic acid (CPA) is reported. The results show that the initial proton-controlled electron transfer (PCET) enables the full conversion of CPA by subsequent C-C coupling and proton transfers.

The characterization and functions of the petJ1 and petJ2 cytochromes isolated from cyanobacterium Synechococcus PCC 7002 are described. The electrostatic and redox properties of the two cytochromes are found to differ radically.

Reduction process of the hydrated uranyl cation, [[U[O.sub.2]].sup.2+], by cyctochromes is studied. A catalytic cycle for the enzymatic reduction of uranyl cation is proposed.

The NMR spectroscopic characterization of the transient complex between plastocycanin (Pc) and cytochrome f (cytf) of the cyanobacterium Prochlorothrix hollandica is described. The mutation of the two hydrophobic patch residues Tyr12 and Pro14 in wild-type Pc-cytf complex provide an opportunity to examine the movements and dynamics in the encounter state of a transient protein-protein complex.

The basic thermodynamic and kinetic parameters for the reaction of cytochrome bd from Azotobacter vinelandii with oxygen was measured and compared with those of the Escherichia coli enzyme. The results suggest that in cytochrome bd, the redox state of the b-type hemes (heme [b.595]), controls the pathway for ligand transfer between heme d and the bulk phase.

The reaction in solution is studied to understand how the very weak binding between the two proteins from Phormidium laminosum can nevertheless lead to fast rates of electron transfer. The results are interpreted in terms of a model in which the stable form of the protein-protein complex requires further thermal activation to be competent for electron transfer.

Electronic structures of iron(II) and iron(III) porphyrins are studied with density functional theory (DFT) using the GGA exchange functional OPTX in combination with the correlation functional PBE (OPBE) and with the correlation functional Perdew (OPedrew) together with a triple zeta-type basis set. These functionals are evaluated against other functionals for their performance in calculating relative energies for the various electronic states of both the iron porphyrins.

The electrochemical reduction of dioxygen is achieved using the BPG-Py-hBM3 system, which is an excellent catalyst. The estimated k(degree) of the system is so high that applying a potential of -0.5 V causes rapid reduction of dioxygen to water.

Evidence is reviewed elucidating the mechanism of iron-induced triggering of antimalarial trioxanes. Several new, orally active antimalarial peroxides were designed rationally based on this fundamental mechanistic paradigm.

Questions with regards to the binding of formate to the binuclear site of bovine cytochrome c oxidase (CcO) by perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) are addressed. Data show unequivocally that formate binds in its anionic form despite its binding being electroneutral overall.

A single crystal of the bovine mitochondrial cytochrome bc1 complex with stigmatellin in the Q (super 0) quinol binding site determines the orientation of the g-tensors of the Rieske iron-sulfur protein subunit. Each principal g-tensor has important implications, for the electronic structure of the 2Fe2S cluster and its role in electron transfer reactions.

Research has been conducted on Ru-diimine complexes binding to cytochrome P450cam. The authors describe the synthesis and characterization of these complexes.

Research has been conducted on ester-containing 5-carboxyl-5-methyl-1-pyrroline N-oxides. The estimated rate constants for these oxides at acidic and neutral pH for the reaction of ester-containing 5-carboxyl-5-methyl-1-pyrroline N-oxides with superoxide have been achieved via the use of ferricytochrome c.

A number of active-site mutations based on a comparison of the structures of camphor and (+)-alpha-pinene are designed and the activity, selectivity and coupling efficiency of these P450(sub cam) mutants for (+)-alpha-pinene oxidation are assayed. The results show that the P450(sub cam)/substrate molecular recognition interactions are both highly dynamic and subtly modulated by amino acid substitutions.

In the study, the first direct spectroscopic evidence for multiple substrates is provided. This evidence is found within a single cytochrome P sub 450 active site.

A study demonstrates that it is possible to successfully display a porphyrin binding protein on an amyloid fibril scaffold and incorporate heme to form a functional b-type cytochrome. The reproductibility and stoichiometric character of the system suggests that further investigations could provide important insights into the nature of the amyloid structure and assembly process.

Result in N-dealkylation and sulfoxidation, two of the most facile reactions mediated by Cytochorme P450 are reported. Results from two experiments are found consistent with sulfoxidation and N-dealkylation rising from two different enzyme substrate complexes (ES) and thus two different active oxygen species that do not interchange.