Oxygen penetration and diffusion into myoglobin revealed by quenching of zincprotoporphyrin IX fluorescence

Carrero, Jenny; Jameson, David M.; Gratton, Enrico

doi:10.1016/0301-4622(94)00133-5

Cited by 7 publications

(7 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the presence of O 2 , the phosphorescence of AZ, LADH and AP is short-lived, and the decay is strictly exponential even for LADH whose intrinsic emission is notoriously heterogeneous. A uniform decay contrasts with O 2 quenching of fluorescence where lifetime heterogeneity has been found to increase at higher quenching efficiency. , Throughout, 1/τ increases linearly with [O 2 ] as should be expected for a dynamic quenching process. Representative lifetime Stern−Volmer plots at 20 °C are shown in Figure , and the bimolecular quenching rate constants, P k q , derived from their slopes are given in Table .…”

Section: Resultsmentioning

confidence: 67%

“…Further, at the 0.1−1 M [O 2 ] needed to quench the fluorescence of inaccessible Trp residues, several O 2 molecules are likely to be always inside the protein, already near the chromophore or within its interaction range. On the other hand, according to a dynamical model advanced to account for fluorescence quenching of chromophores in protein interiors, O 2 migration inside the macromolecule is rapid and occurs freely in the nanosecond time scale, whereas crossing of the water−protein interface is generally opposed by a sizable activation barrier. , The model predicts that F k q > P k q because Trp fluorescence quenching would report on fast internal O 2 diffusion, whereas phosphorescence quenching, which is conducted at μM [O 2 ], would refer to the slow, O 2 entry step. Still another aspect tied to the much smaller [O 2 ] needed to quench phosphorescence is accurate control of [O 2 ] in solution.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Pressure−Temperature Effects on Oxygen Quenching of Protein Phosphorescence

Strambini

Cioni

1999

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Oxygen quenching of protein phosphorescence was determined for the buried Trp residues of apoazurin, liver alcohol dehydrogenase, and alkaline phosphatase as a function of temperature (0-50°C) and applied pressure (up to 3 kbar). Accurate control of the oxygen concentration in solution, by a method that employs an internal protein reference, largely confirms the small bimolecular quenching rate constants (k q ) reported previously for these proteins. Wide variations in flexibility of the globular fold, as attained from protein to protein or by changing external conditions of temperature and pressure, establish that the magnitude of k q is directly correlated to the rigidity of the protein matrix surrounding the chromophore and demonstrates that the quenching rate constant is limited by hindered migration of oxygen through compact regions of the polypeptide. The magnitude of k q implies that O 2 diffusion in proteins can be slowed over 1000-fold relative to water and much more than was inferred from the corresponding fluorescence quenching rate. The activation enthalpy for the structural fluctuations underlying O 2 diffusion in proteins ranges between 9 and 12 kcal mol -1 , similar among the three proteins but larger than the 3 kcal mol -1 for O 2 diffusion in water. The activation volumes, obtained from the pressure dependence of k q , are largest and positive at 50°C and below 2 kbar, but decrease monotonically at higher pressure and at lower temperature. This behavior, together with a similar magnitude of the activation volumes among proteins with different internal mobility are interpreted as to indicate an essential role of internal water molecules in conferring flexibility to protein structure.

show abstract

Section: Resultsmentioning

confidence: 67%

Section: Introductionmentioning

confidence: 99%

Pressure−Temperature Effects on Oxygen Quenching of Protein Phosphorescence

Strambini

Cioni

1999

J. Am. Chem. Soc.

View full text Add to dashboard Cite

show abstract

“…Protein structures derived from both diffraction experiments and nuclear magnetic resonance spectroscopy suggest that some structural flexibility is required for intermolecular access that permits function. For example, myoglobin requires some structural reorganization for oxygen to access the iron atom (Case and Karplus, 1979;Carrero et al, 1995) and ribonuclease does not function below a dynamical transition at 220 K (Rasmssen et al, 1992). Discussions of molecular accessibility are often based on a computational strategy that begins with the highresolution static or dynamically averaged structural model of the protein (Lee and Richards, 1971).…”

Section: Introductionmentioning

confidence: 99%

“…Experimental methods for characterizing intermolecular accessibility include chemical reactivity, amide-hydrogen exchange kinetics (Woodward and Rosenberg, 1971;Woodward et al, 1982;Kim et al, 1993;Wang et al, 1995;Hitchens and Bryant, 1998;Li and Woodward, 1999;Dixon et al, 2000), fluorescence quenching (Lakowicz and Weber, 1973a,b;Calhoun et al, 1983aCalhoun et al, ,b, 1986Gratton et al, 1984;Carrero et al, 1995;Mansoor et al, 1999), electron spin relaxation (ESR) (Altenbach et al, 1989a,b;Victor and Cafiso, 1998), as well as the nuclear magnetic resonance (NMR) contact shifts and relaxation (Esposito et al, 1992;Cocco and Lecomte, 1994;Molinari et al, 1997;Prosser et al, 2001). Fluorescence quenching and ESR experiments, although providing both spatial and dynamic information, suffer from the small number of reporter sites.…”

Section: Introductionmentioning

confidence: 99%

Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

Teng

Bryant

2004

Biophysical Journal

View full text Add to dashboard Cite

Paramagnetic contributions to nuclear magnetic spin-lattice relaxation rate constant induced by freely diffusing molecular oxygen measured at hundreds of different protein proton sites provide a direct means for characterizing the exploration of the protein by oxygen. This report focuses on regions of ribonuclease A where the rate constant enhancements are either quite large or quite small. We find that there are several regions of enhanced oxygen affinity for the protein both on the surface and in interior pockets where sufficient free volume permits. Oxygen has weak associative interactions with a number of surface crevices that are generally between secondary structural elements of the protein fold. Several regions near the surface have higher than expected accessibility to oxygen indicating that structural fluctuations in the protein provide intermolecular access. Oxygen penetrates part of the hydrophobic interior, but affinity does not correlate simply with hydrophobicity indices. Oxygen is excluded from regions of high interior packing density and a few surface sites where x-ray diffraction data have indicated the presence of specific hydration with high occupancy.

show abstract

“…Our data indicate that this site has to be located close enough to the heme to allow such a rebinding to occur within hundreds of picoseconds to nanoseconds. Considering the reported values for CO and oxygen diffusion coefficients within proteins, ranging between 10 Ϫ7 and 10 Ϫ11 cm 2 /s, one can crudely estimate that the distance between the new NO site and the heme should not exceed ϳ10 Å (18,19). Such a distance of ϳ10 Å may be too large to hinder NO recombination to the heme by steric effects.…”

Section: Evidence For a New Non-heme No Binding Site In Enos-mentioning

confidence: 99%

Nitric Oxide (NO) Traffic in Endothelial NO Synthase

Slama‐Schwok¹,

Négrerie²,

Berka³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Nitric oxide (NO) traffic within the reduced ferrousnitrosyl complex of endothelial nitric-oxide synthase (eNOS) has been studied by ultrafast time-resolved absorption spectroscopy. In the presence of tetrahydrobiopterin, the rate of NO rebinding to the heme upon photodissociation depends on the NO concentration. The time scale of this process, picoseconds to nanoseconds, precludes a diffusion from the solution toward the protein medium, and altogether the data point at a new NO binding site within the protein. Comparison of the kinetics of pterin-bound and -depleted eNOS points out that the existence of this new site depends on the presence of tetrahydrobiopterin. The new non-heme site may act as a "doorstep" to the heme pocket and control NO escape from eNOS. Nitric oxide (NO)1 is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric-oxide synthase, eNOS. NO production is crucial in the control of vascular tone, arterial pressure, smooth muscle cell proliferation, and platelet adhesion to the endothelial surface. NOS catalyzes the formation of NO and L-citrulline from L-arginine in the presence of oxygen and NADPH (1). The active protein is a homodimer (2). Upon Ca 2ϩ /calmodulin binding, the FAD of the reductase domain transfers reducing equivalents from NADPH to FMN, which then reduces the heme iron located in the oxygenase domain. This domain also includes binding sites for the substrate, L-arginine, and the cofactor, tetrahydrobiopterin (BH 4 ). The cofactor BH 4 is required for full activity of all NOS isoforms. Reconstitution of holoenzyme activity and BH 4 binding requires the presence of glutathione, necessary to reduce an oxidized cysteine (3). BH 4 binds at the edge of, and perpendicular to, the heme plane through an extensive network of hydrogen bonds (4 -6).In this report, we focus on the effect of NO concentration on the dynamics of NO released from the ferrous-nitrosyl complex, Fe(II)-NO (7), and its potential role in controlling NO release from NOS. The ultrafast rebinding of a dissociated NO molecule to the eNOS heme site, a process called geminate recombination, involves NO molecules that do not escape into the solvent. The kinetics of this rebinding are very sensitive to the environment of the heme. This ligand rebinding is studied at various NO concentrations.In this work, we show that varying the NO to the protein ratio induces changes in the geminate recombination rate for eNOS possessing bound BH 4 . In contrast, the rate of NO rebinding to BH 4 -depleted eNOS was independent of NO concentration and much slower than in the presence of the cofactor. Our data strongly suggest the existence of a new non-heme binding site for NO that depends on the presence of BH 4 . EXPERIMENTAL PROCEDURESMaterials-L-Arginine, ␤-NADPH (99.8% purity), (6R)5,6,7,8-tetrahydrobiopterin (BH 4 ), and calmodulin, as well as other chemicals used for buffer preparations, were purchased from Sigma. Diluted NO gas at 1 and 10% (v/v) in nitrogen and argon, respectively, and high purit...

show abstract

Oxygen penetration and diffusion into myoglobin revealed by quenching of zincprotoporphyrin IX fluorescence

Cited by 7 publications

References 24 publications

Pressure−Temperature Effects on Oxygen Quenching of Protein Phosphorescence

Pressure−Temperature Effects on Oxygen Quenching of Protein Phosphorescence

Mapping Oxygen Accessibility to Ribonuclease A Using High-Resolution NMR Relaxation Spectroscopy

Nitric Oxide (NO) Traffic in Endothelial NO Synthase

Contact Info

Product

Resources

About