Nitrite coordination in myoglobin.

The coordination of nitrite in myoglobin (Mb) has been characterized by resonance Raman spectroscopy and the frequencies of the nitrite bound to the heme Fe as well to the 2-vinyl have been computed by density functional theory (DFT) calculations. The DFT Natural Bond Orbital (NBO) analysis and the extensive isotope-labeling in the resonance Raman experiments indicate that NO2- (O1NO2) is bound to the heme Fe via O1. Based on the vibrational characterization of the reversible transition between low and high spin FeONO/2-nitrovinyl species, we suggest that the key step that triggers the spin-change is the increase of the proximal FeNHis93 bond length. The frequencies of the O and N sensitive bands of the FeONO/2-nitrovinyl species remained largely unchanged in the low- to high-spin transition. Therefore the "greening" process in the reaction of ferric Mb with NO2- proceeds through the FeONO/2-nitrovinyl species, which can exist in either the high or low-spin state.

Coupling of helix E-F motion with the O-nitrito and 2-nitrovinyl coordination in myoglobin.

Myoglobin (Mb) is known to react slowly with nitirite to form the green pigment by NO2- cordination to the heme Fe in the O-binding nitrito (O1NO2) mode and to the heme 2-vinyl position. Nitrite is a powerful oxidizing agent and a biological reservoir for NO that has been implicated in a variety of aerobic biological systems. Accordingly, it is important to elucidate the nature and variety of NO2- reaction mechanisms with Mb. We have performed principal component analysis (PCA, or essential dynamics) on Molecular Dynamics trajectories of all MbNO2 coordination states to resolve the most important motions in the protein at 298K. We show that the coordination or removal of NO2- to/from the heme iron is associated mainly with a motion of helix E and the coordination of NO2- to the 2-vinyl is associated with a motion of helix F and a correlated motion of helices E-F. This latter correlated motion can be attributed to the interaction of Val68 and Ile107 with the 2-nitrovinyl moiety. The resonance Raman results show that coordination of NO2- to the 2-vinyl is increased at pH6.0 demonstrating that the amide protons in the F helix are not protected from access of solvent water and the helix F motion allows solvent access to the 2-vinyl group, without affecting the coordination to the heme Fe.

Spin Crossover in Nitrito-Myoglobin as Revealed by Resonance Raman Spectroscopy.

The myoglobin (Mb) heme Fe-O-N=O and heme Fe-O-N=O/2-nitrovinyl species have been characterized by resonance Raman spectroscopy. In the heme Fe-O-N=O species, the bound nitrite ligand is removed by solvent exchange, thus reforming metmyoglobin (metMb). The high-spin heme Fe-O-N=O unit is converted into a low-spin heme Fe-O-N=O/2-nitrovinyl species that can be reversibly switched between a low- and a high-spin state without removing the bound nitrite ligand, as observed in the case of the heme Fe-O-N=O species. This spin-state change is likely to be accompanied by a general structural rearrangement in the protein-binding pocket. This example is the first of a globin protein that can reversibly change its metal spin state through an internal perturbation. These findings provide a basis for understanding the structure-function relationship of the spin cross found in other metalloenzymes and Fe(III) -porphyrin complexes.

Resonance Raman detection of the myoglobin nitrito heme Fe-O-N=O/2-nitrovinyl species: implications for helix E-helix F interactions.

The description of biological activity in heme proteins responsible for activating small molecules requires identification of ligand movement into the metal and non-metal binding sites. Mechanisms of nitrite reductase activity in globins are difficult to verify without the structures of the bound ligand, but we now have such information from resonance Raman spectroscopy on the myoglobin nitrito heme Fe-O-N=O/2-nitrovinyl species in their natural environment rather than in crystals. Our results indicate that the formation of the nitrito heme Fe-O-N=O/2-nitrovinyl species is pH-dependent. The conditions under which the nitrito heme Fe-O-N=O/2-nitrovinyl species is generated strongly suggest that this form corresponds to an acid induced transformation. We propose that the movement of helices E and F at low pH results in the protonation of nitrito heme Fe-O-N=O by His64 Nε-H(E) to form the nitrous heme Fe-O(H)-N=O species.

The value of adenosine test in the diagnosis of sick sinus syndrome: susceptibility of sinus and atrioventricular node to adenosine in patients with sick sinus syndrome and unexplained syncope.

AIMS: Patients (pts) with sick sinus syndrome (SSS) and unexplained syncope show increased susceptibility of sinus and atrioventricular node (AVN) to intravenous adenosine, respectively. Our aim is to assess the diagnostic value of adenosine test in pts with SSS, as well as to evaluate the response of AVN to adenosine either in pts with unexplained syncope or in pts with syncope and known SSS. METHODS AND RESULTS: The effect of adenosine administration on the sinus and AVN was studied in a population consisted of 19 pts with clinical SSS (group SSS), 7 pts with syncope of unknown origin (group SUO), and 12 control subjects (group C). We calculated the maximum corrected sinus node recovery time (CSNRT), after overdrive pacing of the atrium at cycle lengths of 600, 500, and 400 ms and compared this value with the longest sinus pause, following adenosine administration corrected to the basic cycle length (ADSNRT). The longest R-R interval during atrioventricular block in response to adenosine injection (ADAVB) was also measured. Adenosine was given in a bolus dose of 0.15 mgr/kg through a femoral or large antecubital vein. There was a significant difference in the mean values of CSNRT among the three groups: group SSS (651 +/- 228 ms) > group SUO (284 +/- 100 ms) = group C (291 +/- 117 ms), F(2.35) = 19.078, P = 0.000. A significant difference was also found with ADSNRT: group SSS (5437 +/- 6863 ms) > group SUO (122 +/- 120 ms) = group C (801 +/- 1897 ms), F(2.35) = 4.513, P = 0.018. Using 525 ms as a cutoff value indicating sinus node dysfunction, CSNRT had a sensitivity of 74% and specificity of 100% for diagnosis of SSS while ADSNRT had 94% and 84%, respectively. Higher values of ADAVB in pts with SSS (10659 +/- 5872) and SUO (10026 +/- 7092) in comparison with controls (3615 +/- 5002) were measured, F(2.35) = 5.697, P = 0.007. No difference in the degree of ADAVB was found between the pts with SUO (10026 +/- 7092 ms) and syncope in the presence of SSS (12058 +/- 6787 ms), F(1.15) = 0.356, P = 0.56. CONCLUSION: Adenosine test appears to be at least comparable with CSNRT in making the diagnosis of SSS and may be considered as an alternative non-invasive test for confirmation of suspected SSS. No difference in the susceptibility of AVN to adenosine between the pts with syncope in the presence of SSS and those with unexplained syncope was found, suggesting that adenosine test cannot be used to diagnose atrioventricular block as the cause of syncope.

Brugada type electrocardiographic changes induced by concomitant use of lithium and propafenone in patient with Wolff-Parkinson-White syndrome.

We report a case of ST elevation in right precordial leads compatible with type 1 Brugada syndrome following administration of propafenone in a patient with Wolff-Parkinson-White syndrome who was receiving lithium at concentrations within therapeutic levels. The mechanism of the electrocardiogram changes was considered to be caused by the two drugs synergistic sodium channel blocking effects.