The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp.

Efficient electron transfer in a protein network lacking specific interactions.

In many biochemical processes, proteins need to bind partners amidst a sea of other molecules. Generally, partner selection is achieved by formation of a single-orientation complex with well-defined, short-range interactions. We describe a protein network that functions effectively in a metabolic electron transfer process but lacks such specific interactions.

Amicyanin transfers electrons from methylamine dehydrogenase to cytochrome c-551i via a ping-pong mechanism, not a ternary complex.

The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large.

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers.

The main retinol carriers in the cytosol are the cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II), which exhibit distinct tissue distributions. They play different roles in the maintenance of vitamin A homeostasis and feature a 100-fold difference in retinol affinity whose origin has not been described in detail. NMR-based hydrogen/deuterium exchange measurements show that, while retinol binding endows both proteins with a more rigid structure, many amide protons exchange much faster in CRBP-II than in CRBP-I in both apo and holo form, despite the conserved three-dimensional fold.

A Proton ENDOR Study of Azurin.

As part of our ongoing project that aims at the optimum characterization of the electronic structure of the blue-copper site of azurin from Pseudomonas aeruginosa, we present the complete hyperfine tensors of the protons bound to the Cbeta atom of the copper-bound cysteine 112. These tensors have been obtained from a 95 GHz pulsed electron-nuclear double resonance study of a single crystal of the protein.

Structures of the G81A mutant form of the active chimera of (S)-mandelate dehydrogenase and its complex with two of its substrates.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a membrane-associated flavoenzyme, catalyzes the oxidation of (S)-mandelate to benzoylformate. Previously, the structure of a catalytically similar chimera, MDH-GOX2, rendered soluble by the replacement of its membrane-binding segment with the corresponding segment of glycolate oxidase (GOX), was determined and found to be highly similar to that of GOX except within the substituted segments. Subsequent attempts to cocrystallize MDH-GOX2 with substrate proved unsuccessful.

Redox properties and crystal structures of a Desulfovibrio vulgaris flavodoxin mutant in the monomeric and homodimeric forms.

The mutant S64C of the short-chain flavodoxin from Desulfovibrio vulgaris has been designed to introduce an accessible and reactive group on the protein surface. Crystals have been obtained of both the monomeric and homodimeric forms of the protein, with the cofactor FMN in either the oxidized or the one electron-reduced (semiquinone) state, and the structures have been determined to high resolution. The redox properties of the different species have been investigated and the variations observed with respect to wild type have been related to the structural changes induced by the mutation and S-S bridge formation.

Structural comparison of crystal and solution states of the 138 kDa complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus.

Methylamine can be used as the sole carbon source of certain methylotrophic bacteria. Methylamine dehydrogenase catalyzes the conversion of methylamine into formaldehyde and donates electrons to the electron transfer protein amicyanin. The crystal structure of the complex of methylamine dehydrogenase and amicyanin from Paracoccus versutus has been determined, and the rate of electron transfer from the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase to the copper ion of amicyanin in solution has been determined.

Mass spectrometry and hydrogen/deuterium exchange measurements of alcohol-induced structural changes in cellular retinol-binding protein type I.

To bind and release its ligand, cellular retinol-binding protein type I (CRBP) needs to undergo conformational and dynamic changes to connect the inner, solvent-shielded cavity, where retinol is found to bind, and the outside medium. Retinol dissociation in vitro is favoured by water/alcohol mixtures whose moderately low dielectric constants mimic a property characteristic of the membrane microenvironment where this process occurs in vivo. Apo- and holo-CRBP, in either water/methanol or water/trifluoroethanol (TFE) mixtures, were analyzed at equilibrium by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) to identify the alcohol-induced species.

Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin.

Retinol modulates site-specific mobility of apo-cellular retinol-binding protein to promote ligand binding.

A fundamental question in protein science is how the inherent dynamics of a protein influence its function. If this function involves interactions with a ligand, the protein-ligand encounter has the potential to modulate the protein dynamics. This study reveals how site-specific mobility can be modulated by the ligand to facilitate high affinity binding.

Spin-density distribution in the copper site of azurin.

A 95 GHz pulsed deuterium ENDOR study has been performed on single crystals of azurin from Pseudomonas aeruginosa selectively deuterated at the C(beta) position of the copper-coordinating cysteine 112. Complete hyperfine tensors of the two deuterium atoms have been obtained, which reveal identical isotropic parts. Analysis of the hyperfine tensors provides insight into the spin-density delocalization over the cysteine ligand.

A crystallographic study of Cys69Ala flavodoxin II from Azotobacter vinelandii: structural determinants of redox potential.

Flavodoxin II from Azotobacter vinelandii is a "long-chain" flavodoxin and has one of the lowest E1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 A by molecular replacement.

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein type I.

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation.

Microspectrophotometry for structural enzymology.

For several decades, single-crystal microspectrophotometry has contributed to structural enzymology as a very useful complement to X-ray crystallography. In its most recent applications, it is the ideal tool to track chemistry as structure evolves in the course of time-resolved experiments, to identify freeze-trapped catalytic intermediates and to assess radiation-induced effects on enzyme crystals. To these goals, instruments have been developed to record optical spectra 'on-line' in the course of X-ray data collection, whereas more rigorous polarized absorption studies 'off-line' play an essential role in describing what protein function is retained in the crystalline state and correlating it with the observed structures.

DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.

Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress. The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively. Gel mobility and atomic force microscopy imaging showed that at pH 6.

Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry.

The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure. The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range).

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome c551i as detected by EPR spectroscopy.

EPR studies of the methylamine dehydrogenase (MADH)-amicyanin and MADH-amicyanin-cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry.

Acid-induced denaturation of cellular retinol-binding proteins types I and II studied by electrospray mass spectrometry.

The acid-induced denaturation of cellular retinol-binding proteins types I and II (CRBP I and II), in the presence and in the absence of the ligand, was studied by electrospray ionization mass spectrometry (ESI-MS) in the pH range 6.9-2.4.

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors.

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes.

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.

Comparison of the refined crystal structures of wild-type (1.34 A) flavodoxin from Desulfovibrio vulgaris and the S35C mutant (1.44 A) at 100 K.

Engineered flavodoxins in which a surface residue has been replaced by an exposed cysteine are useful modules to link multi-domain redox proteins obtained by gene fusion to electrode surfaces. In the present work, the crystal structure of the S35C mutant of Desulfovibrio vulgaris flavodoxin in the oxidized state has been determined and compared with a refined structure of the wild type (wt). The structure of wt flavodoxin (space group P4(3)2(1)2, unit-cell parameters a = 50.

Iron incorporation into Escherichia coli Dps gives rise to a ferritin-like microcrystalline core.

Escherichia coli Dps belongs to a family of bacterial stress-induced proteins to protect DNA from oxidative damage. It shares with Listeria innocua ferritin several structural features, such as the quaternary assemblage and the presence of an unusual ferroxidase center. Indeed, it was recently recognized to be able to oxidize and incorporate iron.

Tuning the reduction potential of engineered cytochrome c-553.

Cytochrome c-553 from Desulfovibrio vulgaris exhibits a highly exposed heme and an unusually low reduction potential with respect to other c-type cytochromes. Solvent heme exposure has been indicated as one of the most important factors in modulating the midpoint potential of the redox center. To test this hypothesis, a unique surface-exposed cysteine has been substituted for either M23 or G51 to produce the corresponding mutants and allow the formation of homodimers through a specific disulfide bridge.