Maurizio Brunori

Nitric oxide moves myoglobin centre stage

It has been proposed that myoglobin (Mb), besides being an oxygen carrier, plays the role of a nitric oxide (NO) scavenger in heart and skeletal muscle. A paper reporting data obtained using perfused hearts isolated from either wild-type or Mb-knockout mice provides the first experimental evidence for this novel function of Mb. The biochemical basis underlying the effects of NO on cardiac function is outlined in this article, beginning with the idea that this gas is an inhibitor of cytochrome-c oxidase. Some of the consequences of this new role of Mb and a molecular mechanism to account for the high reactivity of oxymyoglobin with NO are also briefly discussed.

Studies on the functional properties of fish hemoglobins: II. The oxygen equilibrium of the isolated hemoglobin components from trout blood

Homogeneous components of trout hemoglobin (Salmo irideus) have been isolated by column chromatography. The oxygen equilibrium of the two main components has been investigated. The oxygen affinity and the shape of the ligand equilibrium curve is independent of pH for component I. On the other hand, component IV is characterized by a very large Bohr effect, to which a considerable change in the shape of the oxygen equilibrium curve with pH is associated. The different oxygen-binding behavior of the isolated components can explain data obtained with the whole blood and in particular the contribution of the various proteins to the Root effect. The dependence on pH of the apparent ΔH for oxygenation has been measured for both components. Component I is characterized by a fairly low (ΔH ~ −3 kcal/mole) and pH independent value of the enthalpy change, while for component IV the apparent ΔH decreases from ~ −14 kcal/mole at pH near 9 to ~ −7 kcal/mole at pH 7.

Molecular Adaptation to Physiological Requirements: The Hemoglobin System of Trout

Publisher Summary The existence of specific carriers deputed to the transport of oxygen from the outer environment to the place of utilization is widespread in nature. Under the pressure of the variable oxygen requirements, a series of adaptive mechanisms involving the different “gears” of the machine deputed to the supply of oxygen to the tissues has come into operation. Allosteric effects have become apparent in studies on hemoglobins; those on the structural and functional properties of hemoglobins have been largely carried out on the proteins extracted from mammals. Contrary to the case in mammals and birds, a multiplicity of hemoglobin components is generally found in the blood of fish, reptiles, and amphibians. This chapter discusses the structural and functional properties of the various hemoglobin components of the blood of trout ( Salmo irideus ). On the basis of the behavior of the isolated hemoglobin components from trout blood and of their distribution among the erythrocytes, it has been possible to provide a “rationale” for the existence of several hemoglobins.

Nitric oxide, cytochrome-c oxidase and myoglobin

Myoglobin, the monomeric haemoprotein expressed in red muscle, is reported in biochemistry and physiology textbooks to function as an intracellular oxygen carrier and oxygen reservoir. Here, Maurizio Brunori argues that myoglobin can also play the role of intracellular scavenger of nitric oxide, an inhibitor of mitochondrial cytochrome-c oxidase, thereby protecting respiration in the skeletal muscle and the heart.

Complex landscape of protein structural dynamics unveiled by nanosecond Laue crystallography

Although conformational changes are essential for the function of proteins, little is known about their structural dynamics at atomic level resolution. Myoglobin (Mb) is the paradigm to investigate conformational dynamics because it is a simple globular heme protein displaying a photosensitivity of the iron–ligand bond. Upon laser photodissociation of carboxymyoglobin Mb a nonequilibrium population of protein structures is generated that relaxes over a broad time range extending from picoseconds to milliseconds. This process is associated with migration of the ligand to cavities in the matrix and with a reduction in the geminate rebinding rate by several orders of magnitude. Here we report nanosecond time-resolved Laue diffraction data to 1.55-Å resolution on a Mb mutant, which depicts the sequence of structural events associated with this extended relaxation. Motions of the distal E-helix, including the mutated residue Gln-64(E7), and of the CD-turn are found to lag significantly (100–300 ns) behind local rearrangements around the heme such as heme tilting, iron motion out of the heme plane, and swinging of the mutated residue Tyr-29(B10), all of which occur promptly (≤3 ns). Over the same delayed time range, CO is observed to migrate from a cavity distal to the heme known to bind xenon (called Xe4) to another such cavity proximal to the heme (Xe1). We propose that the extended relaxation of the globin moiety reflects reequilibration among conformational substates known to play an essential role in controlling protein function.

The structure of murine neuroglobin: Novel pathways for ligand migration and binding

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 Å reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 Å3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high‐resolution structure, further structure‐function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia. Proteins 2004.

Cavities and packing defects in the structural dynamics of myoglobin

Small globular proteins contain internal cavities and packing defects that reduce thermodynamic stability but seem to play a role in controlling function by defining pathways for the diffusion of the ligand/substrate to the active site. In the case of myoglobin (Mb), a prototype for structure–function relationship studies, the photosensitivity of the adduct of the reduced protein with CO, O2 and NO allows events related to the migration of the ligand through the matrix to be followed. The crystal structures of intermediate states of wild‐type (wt) and mutant Mbs show the photolysed CO to be located either in the distal heme pocket (primary docking site) or in one of two alternative cavities (secondary docking sites) corresponding to packing defects accessible to an atom of xenon. These results convey the general picture that pre‐existing internal cavities are involved in controlling the dynamics and reactivity of the reactions of Mb with O2 and other ligands, including NO.

On the Mechanism of Inhibition of Cytochrome c Oxidase by Nitric Oxide

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a3-CuB binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a3. Reversal of inhibition in the presence of O2 does not involve a direct reaction of O2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s−1), as determined in the absence of O2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a3-CuB center.

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 Å resolution

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.

Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: structural and kinetic aspects.

Schistosomiasis is a parasitic disease affecting over 200 million people currently treated with one drug, praziquantel. A possible drug target is the seleno-protein thioredoxin-glutathione reductase (TGR), a key enzyme in the pathway of the parasite for detoxification of reactive oxygen species. The enzyme is a unique fusion of a glutaredoxin domain with a thioredoxin reductase domain, which contains a selenocysteine (Sec) as the penultimate amino acid. Auranofin (AF), a gold-containing compound already in clinical use as an anti-arthritic drug, has been shown to inhibit TGR and to substantially reduce worm burden in mice. Using x-ray crystallography we solved (at 2.5 Å resolution) the structure of wild type TGR incubated with AF. The electron density maps show that the actual inhibitor is gold, released from AF. Gold is bound at three different sites not directly involving the C-terminal Sec residue; however, because the C terminus in the electron density maps is disordered, we cannot exclude the possibility that gold may also bind to Sec. To investigate the possible role of Sec in the inactivation kinetics, we tested the effect of AF on a model enzyme of the same superfamily, i.e. the naturally Sec-lacking glutathione reductase, and on truncated TGR. We demonstrate that the role of selenium in the onset of inhibition by AF is catalytic and can be mimicked by an external source of selenium (benzeneselenol). Therefore, we propose that Sec mediates the transfer of gold from its ligands in AF to the redox-active Cys couples of TGR.