ATP-dependent conformational dynamics in a photoactivated adenylate cyclase revealed by fluorescence spectroscopy and small-angle X-ray scattering.

Structural insights into the photoactivated adenylate cyclases can be used to develop new ways of controlling cellular cyclic adenosine monophosphate (cAMP) levels for optogenetic and other applications. In this work, we use an integrative approach that combines biophysical and structural biology methods to provide insight on the interaction of adenosine triphosphate (ATP) with the dark-adapted state of the photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata (OaPAC). A moderate affinity of the nucleotide for the enzyme was calculated and the thermodynamic parameters of the interaction have been obtained. Stopped-flow fluorescence spectroscopy and small-angle solution scattering have revealed significant conformational changes in the enzyme, presumably in the adenylate cyclase (AC) domain during the allosteric mechanism of ATP binding to OaPAC with small and large-scale movements observed to the best of our knowledge for the first time in the enzyme in solution upon ATP binding. These results are in line with previously reported drastic conformational changes taking place in several class III AC domains upon nucleotide binding.

Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation.

Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.

Nitric oxide binding to the cardiolipin complex of ferric cytochrome C.

Cardiolipin, a phospholipid specific to the mitochondrion, interacts with the small electron transfer heme protein cytochrome c through both electrostatic and hydrophobic interactions. Once in a complex with cardiolipin, cytochrome c has been shown to undergo a conformational change that leads to the rupture of the bond between the heme iron and the intrinsic sulfur ligand of a methionine residue and to enhance the peroxidatic properties of the protein considered important to its apoptotic activity. Here we report that the ferric cytochrome c/cardiolipin complex binds nitric oxide tightly through a multistep process in which the first step is the relatively slow displacement (5 s(-1)) from heme coordination of an intrinsic ligand that replaces methionine in the complex. Nanosecond photolysis of the nitrosyl adduct demonstrated that a fraction of the nitric oxide escapes from the heme pocket and subsequently recombines to the heme in second-order processes (k = 1.8 × 10(6) and 5.5 × 10(5) M(-1) s(-1)) that, under these conditions, were much faster than recombination of the intrinsic ligand with which they compete. Ultrafast (femtosecond) laser photolysis showed that the geminate recombination of nitric oxide to the heme occurred with time constants (τ = 22 and 72 ps) and that ~23% of the photolyzed nitric oxide escaped into the bulk phase. This high value for the escape fraction relative to other heme proteins indicates the open nature of the heme pocket in this complex. These results are summarized in a scheme and are discussed in terms of the possible modulation of the apoptotic activity of cytochrome c by nitric oxide.

Femtosecond stimulated Raman spectrometer in the 320-520nm range.

Multi-µJ narrow-bandwidth (≈ 10 cm(-1)) picosecond pulses, broadly tunable in the visible-UV range (320-520 nm), are generated by spectral compression of femtosecond pulses emitted by an amplified Ti:sapphire system. Such pulses provide the ideal Raman pump for broadband femtosecond stimulated Raman spectroscopy, as here demonstrated on a heme protein.

Fourier transform infrared investigation of non-heme Fe(III) and Fe(II) decomposition of artemisinin and of a simplified trioxane alcohol.

Fourier transform infrared spectra are reported for the Fe(III)- and Fe(II)-mediated activation of the antimalarial agents artemisinin 1 and its simplified synthetic analogue, trioxane alcohol 2. By monitoring the frequencies of the newly established marker lines in the FTIR spectra, the products of the Fe(II) and Fe(III) reactions have been characterized. In both reactions, artemisinin is activated giving a product mixture of a ring-contracted tetrahydrofuran acetatal 3, C(4)-hydroxy deoxyartemisinin 4, and deoxyartemisinin 5. These data illustrate that the oxidation state of the iron places no restrictions on the endoperoxide reduction mechanism. The FTIR difference (light - dark) spectra indicate that the endoperoxide moiety of artemisinin is photolabile and that the resulted products have the same vibrational characteristics as those observed in the reactions with Fe(II) and Fe(III). The use of 18O-18O enriched endoperoxide in 2 has allowed us to identify two oxygen sensitive modes in the reactions with Fe(II). The reduction of the peroxide bond by Fe(II) in trioxane alcohol 2 follows both the C-C cleavage and 1,5-H shift pathways and produces a ring-contracted tetrahydrofuran acetal 6 which is converted to tetrahydrofuran aldehyde 7 and C(4)-hydroxy deoxytrioxane alcohol 8, respectively. The cleavage of the O-O bond in 1 and 2 by iron and the ability to correlate vibrational properties of the reaction products with structural properties of the isolated products suggest that infrared spectroscopy is an appropriate tool to study the mode of action of antimalarial endoperoxides.

Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin.

Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry.