Gian Luigi Rossi

A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

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 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five‐stranded parallel β‐sheet flanked on either side by α‐helices. An eight‐residue insertion, compared with other long‐chain flavodoxins, forms a short 310 helix preceding the start of the α3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent‐accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E2 value in particular.

Microspectrophotometric measurements on single crystals of coenzyme containing complexes of horse liver alcohol dehydrogenase

In protein crystallography, difference electron density calculations is a wideIy used method to study ligand binding. Interpretation of difference peaks from data to limited resolution gives the position of the ligand on the protein molecule and the rough shape of the bound substance. However, the detailed chemicat nature of the bound molecute often cannot be identified from the difference peaks themselves 6yen at high resolution. For example, structure determinations by difference electron density techniques of dehydrogenase complexes with coenzyme wilt never solve the question in which oxidation state the bound eofactor is present witbJr~ the crystal. The use of electron dense labels on ligands, like Br, C1, and t substituents, frequently has been u~d in ligand Nnding studies in protein crystallography, 8-Br-ADPribose was used in the work with liver alcohol dehydrogenase (LADII) for the identfficatioll of the adenine binding pocket [ I ]. 4-Br-benzaldehyde was introduced as a substrate [2] and the coenzyme analogue 3-I~pyridine adenine dinucteotide was used to demonstrate the binding of an oxidized coenzyme species to the enzyme [3]. Althou~ useful in the X,ray analysis, heavier substituents on ligands might distort the binding mode to the protein. Nonprodue. rive binding can also occur. Obviously, complementary methods of m~alysis of single crystals are necessary when one intends to examine and compare protein structures with naturally occurring cofactors or subst~ates present in different oxidation state. Ill the work with tile flavodoxin semiquinone (radical) structure [4], visible spectra ofsingte cD~tals were recorded [51 which showed that the semiquinone form predominated in the crystals used for X.ray data collection. Structure determinations of LADH complexes have accumulated information about coenzyme analogue binding, inhibitor interactions and conformational changes induced by NADH and substrate [6]. Our goal is to describe in structural terms as many as possible of the individual steps in the process going :from an aldehyde substrate to an alcohot product. In the search for c~stal]ine N,M)Lcontaining complexes the need for additional analytieal methods arose as crystallization experiments on all types of LADH complexes have been performed in an al coholic medium, 4 ;methyl-2,4-pen tanediol (MPD). It seemed especially important to check tbr NADH formation within the single crystals since MPD could act as a substrate during crFstallizatiom Furthermore, in our study of the transient complex between LADH1,4,5,6-tetra.hydronicotinamide adenine dinucleotide (H2NADH) and the chromophoric substrate trans-4-N~Ldimetbylaminocinnamatdehyde (DACA) [71 we needed rNiable tools to measure aldehyde binding within the single c~stal. We report here the use of single crystal microspectrophotometric measurements as a convenient and rapid method to: (I) Detect NADH present ~ the lattice; (2) Follow DACA binding in the active site and O) Test substrate conversion in cD~stals ofcompIexes used for X-ray data collections,

Time course of chemical and structural events in protein crystals measured by microspectrophotometry

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.

Catalytic and regulatory properties of d-glyceraldehyde-3-phosphate dehydrogenase in the crystal: Spectral properties and chemical reactivity of a chromophoric acyl-enzyme intermediate

We have obtained crystals of d -glyceraldehyde-3-phosphate dehydrogenase from the mediterranean lobster Palinurus vulgaris and have investigated the reaction of the crystalline holo-enzyme with the chromophoric acylating reagent β-(2-furyl)acryloyl phosphate by single-crystal microspectrophotometry. As in solution, an acyl-enzyme intermediate builds up, with the stoichiometric limitation of 2·5±0·2 acyl groups per tetramer and with spectral properties and chemical reactivity dependent on binding of NAD+ to the acylated subunits. It is concluded that in the crystal, as well as in solution, specific intersubunit interactions do not allow for simultaneous acylation of the four active sites and that, within the acylated sites, two distinct acyl bond configurations can be attained, corresponding to a catalytically active or inactive enzyme-substrate intermediate. Catalytic deacylation takes place within the crystal, in the presence of NAD+, both via phosphorolysis (or arsenate-assisted hydrolysis) and reduction of the acyl bond by diffusible NADH. Our results show that quaternary structure and active site conformation of the enzyme in the crystal determine the same bonding properties and allow for co-operativity and regulation of catalysis as in solution. This finding argues in favor of the possibility of interpreting the mechanism of d -glyceraldehyde-3-phosphate dehydrogenase on a structural basis.

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. The soil bacterium Paracoccus denitrificans oxidizes a variety of compounds by channeling electrons into the main respiratory pathway. Upon conversion of methylamine by methylamine dehydrogenase, electrons are transported to the terminal oxidase to reduce molecular oxygen. Steady-state kinetic measurements and NMR experiments demonstrate a remarkable number of possibilities for the electron transfer, involving the cupredoxin amicyanin as well as four c-type cytochromes. The observed interactions appear to be governed exclusively by the electrostatic nature of each of the proteins. It is concluded that Paracoccus provides a pool of cytochromes for efficient electron transfer via weak, ill-defined interactions, in contrast with the view that functional biochemical interactions require well-defined molecular interactions. It is proposed that the lack of requirement for specificity in these interactions might facilitate the integration of new metabolic pathways.

Comparison of the refined crystal structures of wild-type (1.34 Å) flavodoxin from Desulfovibrio vulgaris and the S35C mutant (1.44 Å) 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 = 50.52, b = 50.52, c = 138.59 A) at 1.34 A resolution has been refined to R = 0.16 and R(free) = 0.18. The structure of the S35C mutant (space group P4(3)2(1)2, unit-cell parameters a = 50.55, b = 50.55, c = 138.39 A) at 1.44 A resolution has been refined to R = 0.13 and R(free) = 0.16. Data sets were collected with synchrotron radiation at 100 K. In the S35C mutant, the Cys35 thiol group points towards a hydrophobic region, whilst in the wt the Ser35 hydroxyl group points towards a more polar region. The solvent exposure of Cys35 is 43 A(2), of which 8 A(2) is for the sulfur. This is comparable to the exposure of 48 A(2) found for the wt Ser35, where that of the hydroxyl oxygen is also 8 A(2).

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. We studied the interactions between these proteins in solution. A titration followed by NMR spectroscopy shows that amicyanin binds cytochrome c-551i. The interface comprises the hydrophobic and positive patches of amicyanin, not the binding site observed in the ternary complex. NMR experiments further show that amicyanin binds tightly to MADH with an interface that matches the one observed in the crystal structure and that mostly overlaps with the binding site for cytochrome c-551i. Upon addition of cytochrome c-551i, no changes in the NMR spectrum of MADH-bound amicyanin are observed, suggesting that a possible interaction of the cytochrome with the binary complex must be very weak, with a dissociation constant higher than 2 mM. Reconstitution of the entire redox chain in vitro demonstrates that amicyanin can react rapidly with cytochrome c-551i, but that association of amicyanin with MADH inhibits this reaction. It is concluded that electron transfer from MADH to cytochrome c-551i does not involve a ternary complex but occurs via a ping-pong mechanism in which amicyanin uses the same interface for the reactions with MADH and cytochrome c-551i.

Biological activity in the crystalline state

Abstract Proteins in the crystalline state retain biological activity although lattice constraints, the composition of the bathing medium and the low temperatures required for the stabilization of catalytic intermediates might alter their structure and dynamics with respect to the solution phase. Detailed functional studies are essential to the planning and the interpretation of time-resolved X-ray crystallographic experiments.

Light and gtp effects on the turbidity of frog visual membrane suspensions

SummaryThe time course of turbidity changes of frog visual membranes, dependent on osmotic shocks, on light and on nucleotide substrates or effectors of enzyme activities, were measured as absorption changes in a rapid mixing stopped-flow spectrophotometer.As a result of studies on different preparations, it is concluded that light can cause both rapid (within 50 msec) and slow (within 90 sec) changes in the turbidity of visual membranes, not associated with permeability changes, and that they are affected by GTP or its analog guanyl-5′-yl imidodiphosphate; however, the light and GTP effects are lost when a water soluble fraction containing the light-sensitive enzyme cGMP-phosphodiesterase, is removed from the rod outer segments membranes.It is suggested that the fast light and GTP-sensitive response is related to the activation of cGMP-phosphodiesterase.