Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles.

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme

Elucidating molecular connetion between IAHSP onset and Alsin protein by means of Homology Modelling and Molecular Dynamics

The Infantile-onset Ascending Hereditary Spastic Paralysis (IAHSP) is an incurable rare neurodegerative disease related to a mutation-driven aberrant behaviour of the Alsin protein. The lack of information on Alsin atomic structure limits a complete understanding on pathology mechanisms. In this work, molecular modelling techniques have been applied to shed lights on Alsin folding dynamics and misfunction induced by aberrant mutations

Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant "Gly-loop", the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs

Subcellular localization of the five members of the human steroid 5α-reductase family

In humans the steroid 5a-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: D4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization. We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5a-reductase family as both N- and Cterminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates

Dissecting the cytochrome P450 OleP substrate specificity: evidence for a preferential substrate

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound

ALS2-Related Motor Neuron Diseases: From Symptoms to Molecules

Infantile-onset Ascending Hereditary Spastic Paralysis, Juvenile Primary Lateral Sclerosis and Juvenile Amyotrophic Lateral Sclerosis are all motor neuron diseases related to mutations on the ALS2 gene, encoding for a 1657 amino acids protein named Alsin. This ~185 kDa multi-domain protein is ubiquitously expressed in various human tissues, mostly in the brain and the spinal cord. Several investigations have indicated how mutations within Alsin’s structured domains may be responsible for the alteration of Alsin’s native oligomerization state or Alsin’s propensity to interact with protein partners. In this review paper, we propose a description of differences and similarities characterizing the above-mentioned ALS2-related rare neurodegenerative disorders, pointing attention to the effects of ALS2 mutation from molecule to organ and at the system level. Known cases were collected through a literature review and rationalized to deeply elucidate the neurodegenerative clinical outcomes as consequences of ALS2 mutation

Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs

Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin

Visible light can break the Fe—CO bond in Fe(II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T < 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T = 20 K) of an oriented single crystal (0.2 × 0.2 × 0.3 mm) of sperm whale MbCO (space group P21) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle α = 23°) or perpendicular (α = 86°) to the heme normal of each myoglobin molecule. The crystal was continuously illuminated by a white-light source during the data collection. The polarized data give novel information on the Fe-heme electronic/structural rearrangement following photolysis. The XANES (X-ray absorption near-edge structure) spectrum polarized in the direction close to the Fe—CO bond changes dramatically after photolysis, exhibiting a shift of ∼2 eV, due to electronic relaxation of empty states of pz symmetry, while more subtle changes are observed in the spectrum polarized along the heme plane, sensitive to the heme-plane geometry. Changes in the pre-edge region can be interpreted to provide insight into the electronic structure of the highest occupied and lowest unoccupied molecular orbitals (HOMO–LUMO) in the MbCO → Mb* photochemical reaction at low temperature

Time-resolved crystallography for protein structure: the case of heme proteins

Time-resolved Laue diffraction is an elegant approach by which polychromatic X-ray pulses are used in combination with rapid laser triggering of light-sensitive samples so as to make a movie of protein reaction dynamics in real time. The current state-of-the-art with protein Laue diffraction has been time-resolved studies of light-induced conformational changes in myoglobin-CO with 150 ps resolution. Myoglobin has traversed the whole history of pump–probe Laue diffraction, yielding a massive amount of data that have provided considerable insight into the understanding of protein dynamics. A picture of factors governing myoglobin dynamics following ligand dissociation has been drawn from data arising from wild-type and mutant proteins. Other heme proteins have been studied using this approach: Scapharca inequivalvis dimeric hemoglobin, the FixL PAS domain and the photosynthetic reaction center complex of Blastochloris viridis