Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond–nanosecond pump–probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M−1s−1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s−1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus of NP7, a member of the NO transporter nitrophorin family, contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7

Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

Preparing for SwissFEL: Exploring the limits of time-resolved X-ray spectroscopy

Using x-ray absorption spectroscopy (XAS) and x-ray emission spectroscopy (XES) to probe laser-excited samples we can obtain excited-state structural and electronic dynamical information not available through other techniques.[1] One of the restrictions of most synchrotron-based implementations of this technique is the three-orders of magnitude mismatch between x-ray and laser repetition rates (MHz Vs. kHz). By using a laser capable of generating significant pulse energies at MHz repetition rates we can eliminate this discrepancy, thus greatly reducing measurement times and enhancing achievable signal-to-noise ratios.[2] Several examples that demonstrate the strength of this technique will be presented, including probing the electron relaxation in photoexcited TiO2 nanoparticles in solution and resolving the dynamical structural changes associated with ligand rebinding in the protein myoglobin under physiological conditions. The ability to extend these types of measurements into the femtosecond regime at Experimental Station A[3] at the SwissFEL hard X-ray free electron laser, which is under construction at the Paul Scherrer Institute (Villigen, Switzerland), will be discussed.