Raymond M. Esquerra

Water and ligand entry in myoglobin: Assessing the speed and extent of heme pocket hydration after CO photodissociation

A previously undescribed spectrokinetic assay for the entry of water into the distal heme pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%, 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and H2O entry into the empty distal pocket of myoglobin are markedly different, 8 × 107 and 2 × 105 M–1·s–1, respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry.

Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics

The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the alpha,beta chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, n(w), with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native alpha chains than in the beta chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin.

The pH Dependence of Heme Pocket Hydration and Ligand Rebinding Kinetics in Photodissociated Carbonmonoxymyoglobin

We monitored the occupancy of a functionally important non-coordinated water molecule in the distal heme pocket of sperm whale myoglobin over the pH range 4.3-9.4. Water occupancy was assessed by using time-resolved spectroscopy to detect the perturbation of the heme visible band absorption spectrum caused by water entry after CO photodissociation ( Goldbeck, R. A., Bhaskaran, S., Ortega, C., Mendoza, J. L., Olson, J. S., Soman, J., Kliger, D. S., and Esquerra, R. M. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1254-1259 ). We found that the water occupancy observed during the time interval between ligand photolysis and diffusive recombination decreased by nearly 20% as the pH was lowered below 6. This decrease accounted for most of the concomitant increase in the observed CO bimolecular recombination rate constant, as the lower water occupancy presented a smaller kinetic barrier to CO entry into the pocket at lower pH. These results were consistent with a model in which the distal histidine, which stabilizes the water molecule within the distal pocket by accepting a hydrogen bond, tends to swing out of the pocket upon protonation and destabilize the water occupancy at low pH. Extrapolation of this model to lower pH suggests that the additional increase in ligand association rate constant observed previously in stopped-flow studies at pH 3 may also be due in part to reduced distal water occupancy concomitant with further His64 protonation and coupled protein conformational change.

Optical Detection of Disordered Water within a Protein Cavity

Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by X-ray crystallography. Here, we show that water can be detected within the distal cavities of myoglobin mutants by subtle changes in the absorbance spectrum of pentacoordinate heme, even when the presence of solvent is not readily observed in the corresponding crystal structures. A well-defined, noncoordinated water molecule hydrogen bonded to the distal histidine (His64) is seen within the distal heme pocket in the crystal structure of wild type (wt) deoxymyoglobin. Displacement of this water decreases the rate of ligand entry into wt Mb, and we have shown previously that the entry of this water is readily detected optically after laser photolysis of MbCO complexes. However, for L29F and V68L Mb no discrete positions for solvent molecules are seen in the electron density maps of the crystal structures even though His64 is still present and slow rates of ligand binding indicative of internal water are observed. In contrast, time-resolved perturbations of the visible absorption bands of L29F and V68L deoxyMb generated after laser photolysis detect the entry and significant occupancy of water within the distal pockets of these variants. Thus, the spectral perturbation of pentacoordinate heme offers a potentially robust system for measuring nonspecific hydration of the active sites of heme proteins.

Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin

Water molecules can enter the heme pockets of unliganded myoglobins and hemoglobins, hydrogen bond with the distal histidine, and introduce steric barriers to ligand binding. The spectrokinetics of photodissociated CO complexes of human hemoglobin and its isolated α and β chains were analyzed for the effect of heme hydration on ligand rebinding. A strong coupling was observed between heme hydration and quaternary state. This coupling may contribute significantly to the 20-60-fold difference between the R- and T-state bimolecular CO binding rate constants and thus to the modulation of ligand reactivity that is the hallmark of hemoglobin allostery. Heme hydration proceeded over the course of several kinetic phases in the tetramer, including the R to T quaternary transition. An initial 150 ns hydration phase increased the R-state distal pocket water occupancy, nw(R), to a level similar to that of the isolated α (∼60%) and β (∼10%) chains, resulting in a modest barrier to ligand binding. A subsequent phase, concurrent with the first step of the R → T transition, further increased the level of heme hydration, increasing the barrier. The final phase, concurrent with the final step of the allosteric transition, brought the water occupancy of the T-state tetramer, nw(T), even higher and close to full occupancy in both the α and β subunits (∼90%). This hydration level could present an even larger barrier to ligand binding and contribute significantly to the lower iron reactivity of the T state toward CO.

Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism Measurements

A proteins folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity-the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cells heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli (1) provide a measurement of myoglobin mobility in the cytoplasm, (2) taken together with in vitro TRLD studies yield new insights into the nature of the cytoplasmic environment in cells, and (3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.

Abstract 5111: Nitric oxide and its role in photodynamic therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Porphyrinic pigments are used as photosensitizers (PS) in photodynamic detection (PDD) and therapy (PDT) that is a minimally invasive modality in the fight against cancer. When the PS is activated by visible light at a given wavelength, reactive oxygen species (ROS) are generated, which cause cancer cells to undergo cell death. Despite significant advances, drawbacks of the PSs in clinical use include their non-selectivity in cellular-targeting causing cell death by necrosis leading to tissue inflammation. Nitric oxide (NO) has been shown to play a key role in modulating apoptotic cell death pathways and to react with reactive oxygen species to form additional lethal reactive nitrogen species (RNS). In our efforts to enhance the effectiveness of PDT, we set out to investigate the role of NO in PDT. We hypothesized that NO delivered to cancer cells at the time that the PS was administered would enhance the efficacy of PDT by promoting mitochondria-mediated apoptosis. To this end, we incubated androgen-sensitive human prostate adenocarcinoma (LNCaP) cells with both a PS and an NO releasing agent that was most effective in NO release as was spectrophotometrically determined by its oxidation of hemoglobin. Phototoxicity experiments were carried out at 37 OC with a noncoherent light source. Cell viability, damage and death were assessed in both illuminated and non-illuminated cells and were quantified by MTT staining as well as trypan blue and propidium iodide exclusion. To corroborate the cell viability results, we assayed clonogenic recovery in response to PDT in pigmented cells both in the presence and absence of NO. Our results indicate that the effectiveness of PDT in causing cell death depends on the NO concentration. PDT with NO alone was toxic to the cancer cells at high concentration of NO, whereas at low NO concentration no significant cell damage was observed after light illumination. PDT with the PS alone was not as effective in promoting cell death as PDT in the presence of both NO and the PS. Depending on the concentrations of the PS and NO, we observed that either necrosis or apoptosis were the prevailing modes of cell death after PDT. Our results indicate that the phototoxicity of the compounds is mainly determined by their intracellular concentration. Thus, understanding the combined effects of NO and the PSs in enhancing cell phototoxicity will aid in determining the roles that NO plays in improving the efficacy of PDT and may provide an alternate regimen to enhance PDT efficacy. Citation Format: Marco Monroy, Pooncharas Tipgunlakant, Ursula Simonis, Raymond Esquerra. Nitric oxide and its role in photodynamic therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2014-5111