Jayashree Soman

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.

Blocking the gate to ligand entry in human hemoglobin.

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s−1 in α subunits and ∼10-fold faster in β subunits, ∼175,000 s−1. Crystal structures were determined for tetrameric α(WT)β(Trp-63) HbCO, α(Trp-58)β(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) βCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k′CO, to the open conformations of both mutant Hb subunits is ∼80–90 μm−1 s−1, whereas k′CO for the completely closed states is 1000-fold slower, ∼0.08 μm−1 s−1. A transient intermediate with k′CO ≈ 0.7 μm−1 s−1 is observed after photolysis of Trp-63(E7) βCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and βCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.

Determination of Ligand Pathways in Globins APOLAR TUNNELS VERSUS POLAR GATES

Background: O2 pathways in animal hemoglobins and myoglobins are controversial. Results: Ligands enter and exit sperm whale Mb and Cerebratulus lacteus Hb by completely different pathways. Conclusion: Rational mutagenesis mapping can identify ligand migration pathways and provides experimental benchmarks for testing molecular dynamics simulations. Significance: Globins can use either a polar gate or an apolar tunnel for ligand entry. Although molecular dynamics simulations suggest multiple interior pathways for O2 entry into and exit from globins, most experiments indicate well defined single pathways. In 2001, we highlighted the effects of large-to-small amino acid replacements on rates for ligand entry and exit onto the three-dimensional structure of sperm whale myoglobin. The resultant map argued strongly for ligand movement through a short channel from the heme iron to solvent that is gated by the distal histidine (His-64(E7)) near the solvent edge of the porphyrin ring. In this work, we have applied the same mutagenesis mapping strategy to the neuronal mini-hemoglobin from Cerebratulus lacteus (CerHb), which has a large internal tunnel from the heme iron to the C-terminal ends of the E and H helices, a direction that is 180° opposite to the E7 channel. Detailed comparisons of the new CerHb map with expanded results for Mb show unambiguously that the dominant (>90%) ligand pathway in CerHb is through the internal tunnel, and the major (>75%) ligand pathway in Mb is through the E7 gate. These results demonstrate that: 1) mutagenesis mapping can identify internal pathways when they exist; 2) molecular dynamics simulations need to be refined to address discrepancies with experimental observations; and 3) alternative pathways have evolved in globins to meet specific physiological demands.

Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

We present a crystallography chip enabling in situ room temperature crystallography at microfocus synchrotron beamlines and X-ray free-electron laser (X-FEL) sources. Compared to other in situ approaches, we observe extremely low background and high diffraction data quality. The chip design is robust and allows fast and efficient loading of thousands of small crystals. The ability to load a large number of protein crystals, at room temperature and with high efficiency, into prescribed positions enables high throughput automated serial crystallography with microfocus synchrotron beamlines. In addition, we demonstrate the application of this chip for femtosecond time-resolved serial crystallography at the Linac Coherent Light Source (LCLS, Menlo Park, California, USA). The chip concept enables multiple images to be acquired from each crystal, allowing differential detection of changes in diffraction intensities in order to obtain high signal-to-noise and fully exploit the time resolution capabilities of XFELs.

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.

Post-translational Transformation of Methionine to Aspartate Is Catalyzed by Heme Iron and Driven by Peroxide A NOVEL SUBUNIT-SPECIFIC MECHANISM IN HEMOGLOBIN

Background: Met(E11) mutation in Hb heme pocket undergoes modification to Asp specifically in γ and β subunits. Results: Asp conversion increased in β/γ chains with increasing peroxide and was seen after autoxidation of mutant hemoglobin crystals. Conclusion: Asp is formed by an oxidative mechanism involving ferryl heme complexes. Significance: Subunit-specific modifications aid in the interpretation of hemoglobinopathies and design of oxidatively stable hemoglobins. A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and β subunits of adult Hb (HbA) Bristol-Alesha (β67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H218O2, we verified incorporation of 18O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in β subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, β, and γ subunits with respect to redox reactivity and may have direct implications to α/β hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

Conformational variation of calcium-bound troponin C.

Introduction. Troponin is the Ca21-binding regulatory protein in mammalian skeletal and cardiac muscles. Together with tropomyosin, troponin regulates the interaction between myosin crossbridges and actin in response to rising and falling levels of intracellular Ca21 (1025 to 1027 M). The troponin complex has three subunits: troponin C (TnC) which binds Ca21 specifically, troponin I (TnI), and troponin T (TnT). When TnC binds 4 Ca21 ions, it undergoes a conformational transition which is transmitted to TnI, causing TnI to release its inhibition of the actinmyosin interaction via TnT and tropomyosin.1 TnC contains four Ca21-binding sites: two high-affinity sites in the C-domain that also bind Mg21 and are always occupied, and two low-affinity Ca21-specific sites in the N-domain. Based on a wealth of studies, including the crystal structure of 2-Ca21 avian TnC2,3 and the NMR structure of calcium-saturated TnC,4 it is generally accepted now that muscle contraction is initiated by Ca21binding to the N-domain, causing the domain to undergo a transition from a closed conformation to an open one and exposing a hydrophobic patch. It has been suggested that a region of TnI may then bind to this patch. The recent crystal structures of 2-Ca21chicken N-TnC5 and 4-Ca21 rabbit TnC6 corroborate the conformational transition in the N-domain. We present here the crystal structure of 4-Ca21 rabbit TnC in a new crystal form. A detailed comparison with other calcium-saturated structures shows differences that help describe the dynamics of TnC and its range of conformations.

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.

Thermoglobin, Oxygen-avid Hemoglobin in a Bacterial Hyperthermophile

The hemoglobin family of proteins, ubiquitous in all domains of life, evolved from an ancestral protein of primordial function to extant hemoglobins that perform a myriad of functions with diverged biochemical properties. Study of homologs in bacterial hyperthermophiles may shed light on both mechanisms of adaptation to extreme conditions and the nature of the ancestral protein. A hemoglobin was identified in Aquifex aeolicus, cloned, recombinantly expressed, purified, and characterized. This hemoglobin is monomeric, resistant to thermal and chemical denaturation, pentacoordinate in the ferrous deoxygenated state, and oxygen-avid. The oxygen equilibrium dissociation constant is approximately 1 nm at room temperature, due in part to a hydrogen bond between the bound ligand and a tyrosine residue in the distal pocket. These biochemical properties of A. aeolicus thermoglobin, AaTgb, may have been shared by the ancestral hemoglobin, thus suggesting possible primordial functions and providing a starting point for consequent evolution of the hemoglobin family.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Fe Deficiency and Increased CO Binding.

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin α chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) → Leu mutation in α1. Surprisingly, the patients father is not anemic, but he is a smoker with high levels of HbCO (∼16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the α H58L replacement. Mutant α subunits containing Leu-58(E7) autoxidize ∼8 times and lose hemin ∼200 times more rapidly than native α subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the α H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant α subunit has an ∼80,000-fold higher affinity for CO than O2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.