and A. Grant Mauk

Side-On Copper-Nitrosyl Coordination by Nitrite Reductase

A copper-nitrosyl intermediate forms during the catalytic cycle of nitrite reductase, the enzyme that mediates the committed step in bacterial denitrification. The crystal structure of a type 2 copper-nitrosyl complex of nitrite reductase reveals an unprecedented side-on binding mode in which the nitrogen and oxygen atoms are nearly equidistant from the copper cofactor. Comparison of this structure with a refined nitrite-bound crystal structure explains how coordination can change between copper-oxygen and copper-nitrogen during catalysis. The side-on copper-nitrosyl in nitrite reductase expands the possibilities for nitric oxide interactions in copper proteins such as superoxide dismutase and prions.

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Dissociation of Heme from Myoglobin and Cytochrome b5: Comparison of Behavior in Solution and the Gas Phase†,‡

The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme-protein complexes in the orifice-skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabilized in solution but stabilized in the gas-phase ions. These results suggest that on the millisecond time scale of the ES-MS experiment, the gas-phase protein ion may retain much of the structure of the protein in solution, at least for those residues surrounding the heme group. Furthermore, the ability of ES-MS to detect relatively subtle differences in protein-small molecule complex stability demonstrated in this work suggests that this technique may be a convenient, sensitive, and generally useful strategy for physical characterization of such complexes.

Reaction of human myoglobin and H2O2. Involvement of a thiyl radical produced at cysteine 110.

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H2O2(protein/H2O2 = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO⋅) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g ∼ 2.036) was detected after decay of Trp-OO⋅ that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios ≤1:10 mol/mol, a DMPO adduct exhibiting broad absorptions was detected. Hyperfine couplings of this radical indicated a DMPO-thiyl radical. Incubation of wild-type human Mb with thiol-blocking reagents prior to reaction with peroxide inhibited DMPO adduct formation, whereas at protein/DMPO ratios ≥1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H2O2demonstrated the formation of a homodimer (mass of 34,107 ± 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H2O2 differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.

Structural and spectroscopic studies of azide complexes of horse heart myoglobin and the His-64-->Thr variant.

The high-resolution X-ray crystallographic structures of horse heart azidometmyoglobin complexes of the wild-type protein and the His-64-->Thr variant have been determined to 2.0 and 1.8 A respectively. Azide binds to wild-type metmyoglobin in a bent configuration with an Fe-N-1-N-3 angle of 119 degrees and is oriented into the distal crevice in the direction of Ile-107. The proximity of the His-64 NE2 atom to the N-1 atom of the bound azide indicates stabilization of the ligand by the His-64 side chain through hydrogen bonding. In addition, structural characterization of wild-type horse heart azidometmyoglobin establishes that the only structural change induced by ligand binding is a small movement of the Leu-29 side chain away from the azide ligand. EPR and Fourier transform infrared spectroscopy were used to characterize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly different low-spin species are formed by azide bound to the His-64-->Thr variant both in solution and in a polycrystalline sample. One of these low-spin species has a greater relative intensity, with g values very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indicate the presence of two distinct conformations of bound azide, with one form predominating. The major conformation is comparable to that formed by wild-type myoglobin in which azide is oriented into the distal crevice. In the minor conformation the azide is oriented towards the exterior of the protein.

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1H NMR spectroscopy indicates that Mn2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn2+ is 1.28(4) × 104 M−1 (pH 6.96, ionic strength I = 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pKa for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn2+-peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn2+. The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn2+ and Cd2+. Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

Role of Rhodobacter sphaeroides Photosynthetic Reaction Center Residue M214 in the Composition, Absorbance Properties, and Conformations of HA and BA Cofactors

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

NMR Studies of Paramagnetic Systems to Characterise Small Molecule:Protein and Protein:Protein Interactions

Intermolecular interactions are a feature of all biochemical processes, and whilst some involve the interaction of small molecules with biopolymers or membranes, others involve the interaction of two or more biopolymers. In all cases, a thorough description of the molecular details of the interaction requires kinetic, thermodynamic and structural investigations. In general, however, there are a limited number of possible kinetic and thermodynamic schemes.