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Plant hemoglobins constitute three distinct groups: symbiotic, nonsymbiotic, and truncated hemoglobins. Structural investigation of symbiotic and nonsymbiotic (class I) hemoglobins revealed the presence of a vertebrate-like 3/3 globin fold in these proteins. In contrast, plant truncated hemoglobins are similar to bacterial truncated hemoglobins with a putative 2/2 α-helical globin fold. While multiple structures have been reported for plant hemoglobins of the first two categories, for plant truncated globins only one structure has been reported of late. Here, we report yet another crystal structure of the truncated hemoglobin from Arabidopsis thaliana (AHb3) with two water molecules in the heme pocket, of which one is distinctly coordinated to the heme iron, unlike the only available crystal structure of AHb3 with a hydroxyl ligand. AHb3 was monomeric in its crystallographic asymmetric unit; however, dimer was evident in the crystallographic symmetry, and the globin indeed existed as a stable dimer in solution. The tertiary structure of the protein exhibited a bacterial-like 2/2 α-helical globin fold with an additional N-terminal α-helical extension and disordered C-termini. To address the role of these extended termini in AHb3, which is yet unknown, N- and C-terminal deletion mutants were created and characterized and molecular dynamics simulations performed. The C-terminal deletion had an insignificant effect on most properties but perturbed the dimeric equilibrium of AHb3 and significantly influenced azide binding kinetics in the ferric state. These results along with the disordered nature of the C-terminus indicated its putative role in intramolecular or intermolecular interactions probably regulating protein-ligand and protein-protein interactions. While the N-terminal deletion did not change the overall globin fold, stability, or ligand binding kinetics, it seemed to have influenced coordination at the heme iron, the hydration status of the active site, and the quaternary structure of AHb3. Evidence indicated that the N-terminus is the predominant factor regulating the quaternary interaction appropriate to physiological requirements, dynamics of the side chains in the heme pocket, and tunnel organization in the protein matrix.

Trajectories of diatomic ligands in heme proteins have provided a wealth of information on how such molecules can enter and exit the protein, which must fluctuate in order to permit their passage. The time-resolved X-ray crystallography technique has provided the most detailed images of the transport of the carbon monoxide ligand (CO) to various cavities within proteins (1–5). However, the function of protein cavities is still an open question. Indeed, it is difficult to assign a general function based on the analysis of a single protein, since the functional role of cavities may vary from one protein to another. Thus far, two heme proteins, Sperm Whale myoglobin (SWMb) (2–3) and Scapharca inaequivalvis dimeric hemoglobin (HbI) (4–5), have been the most extensively studied by time-resolved crystallography. Although one is a myoglobin and the other a hemoglobin they both have the reversible binding of O2 as their primary function. Despite the rich set of conformational changes associated with the allosteric cooperative transition in photolyzed HbI*CO, the nature of the CO trajectory in that globin has certain similarities with SWMb*CO (The asterisk indicates a photolyzed carbon monoxide molecule, following photon absorption by the heme that leads to rupture of the Fe-CO bond). Both have a distal docking site B, where the initially photolyzed CO molecule resides. The B-site in both proteins is a distinct ligand docking site in the distal pocket, not observed as the Xe binding site in either proteins. (2, 4) Based on experiments designed to block the Xe-binding cavities in SWMb*CO and HbI*CO, and on time-resolved crystallographic studies, it has been deduced that the cavities in these proteins do not constitute an exit route for diatomic ligands (6–7). Instead, exit occurs near the distal histidine in both SWMb*CO and HbI*CO. Moreover, previous temperature derivative spectroscopy (TDS) studies show the presence of a nearby docking site and one or more further secondary sites in SWMb (8–10) and HbI (11) where photolyzed CO molecules can reside. Why then does CO migrate to a number of Xe-binding cavities in these proteins after it leaves the B-site? This fundamental question has been studied extensively in SWMb using many spectroscopic techniques including TDS and methods such as kinetic hole burning (12), which makes the connection between the conformation-dependent energy of recombination and the spectroscopic energy of a heme charge transfer band. In this study we propose that in order to understand the possible function of the cavities in various heme proteins, it is necessary to compare a protein with a significant difference in function. We conclude that ligand dynamics studied by time-resolved X-ray and the protein cavity in DHP identified by a Xe binding site (13) are consistent with an open architecture in the distal pocket of the bifunctional hemoglobin, DHP, which explains how it can carry out multiple functions. These features distinguish DHP from the more specialized oxygen transport proteins SWMb and HbI. Dehaloperoxidase-hemoglobin (DHP), first isolated from the terebellid polychaete Amphitrite ornata, is a bifunctional protein, which displays significant peroxidase activity under physiological conditions, while also having a globin fold and an associated oxygen transport function (14). As a hemoglobin, DHP can reversibly bind with O2 and as a peroxidase, DHP can oxidize 2,4,6-trihalophenol into the corresponding 2,6-dihaloquinone in the presence of H2O2. However, normal peroxidase function inactivates the ability of DHP to bind oxygen leading to a paradox because of the failure of the DHP hemoglobin function. (15) Recently, this functional paradox of DHP has been resolved by establishing that oxyferrous DHP can serve as the resting state of the peroxidase reaction cycle. (16–17) For a typical peroxidase, e.g. Horseradish Peroxidase and Cytochrome c Peroxidase, the ferric state of the heme Fe is the resting state. Thus, DHP appears to have a new type of peroxidase cycle, a ferrous peroxidase cycle. (15) Although it has high peroxidase activity, the oxyferrous peroxidase cycle and unique internal binding sites possessed by DHP distinguish it from other heme peroxidases. (18–19) The replacement of O2 bound to the heme Fe by H2O2 required by the ferrous peroxidase mechanism suggests DHP must simultaneously accommodate both O2 and H2O2 in the distal pocket in order to function. This requirement is quite different from other well-studied hemoglobins and myoglobins. For example, SWMb, which is by far the best studied protein, cannot simultaneously accommodate both CO and H2O in the distal pocket (20–22). The reasons for this may become clearer in comparison with the bifunctional protein, DHP, which has a different requirement for the function of the distal pocket, namely entry of multiple molecules both for activation and for inhibition of enzymatic function. In order to address the dynamics of diatomic ligands in DHP, we have conducted a time-resolved X-ray crystallography study of the trajectory of photolyzed carbon monoxide in DHP*CO. The time-dependent structures reveal a qualitatively different CO trajectory than those observed previously in SWMb*CO and HbI*CO.

A binding site for iron protoporphyrin IX (heme) was designed and embedded in a photosynthetic non-heme protein, phycocyanin, which forms a globin-like backbone structure, called a globin fold, but lacks sequence similarity to the globin family containing myoglobins and hemoglobins. Based on the structural alignment of the phycocyanin and myoglobin molecules, the proximal and distal His residues were repositioned in the phycocyanin sequence for heme ligation. The heme-binding pocket was created around the His residues by several residue replacements in the phycocyanin core. The synthesized phycocyanin variant, designated as HPY, bound one heme per protein molecule and showed spectroscopic features characteristic of six-coordinated heme proteins. The heme-binding HPY exhibited redox activity with an electrochemical midpoint potential of -130 mV against the standard hydrogen electrode, which was approximately 200 mV lower than the potential of natural myoglobins but 50 mV higher than the typical values of designed heme proteins with four-helix bundle or globin scaffolds. HPY also displayed native-like folding properties, in contrast to these designed heme proteins. However, the bound ferrous heme of HPY was quickly reoxidized by air and did not stably bind O(2), unlike the natural globins. The present results demonstrated that the globin fold of a non-globin protein is suitable for binding heme but is not sufficient for the reversible O(2) binding and myoglobin functions. The comparison of HPY with the natural globins may yield new insights into the essential features for realizing the natural heme protein functions.

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.

Myoglobin was reconstituted with 6,7-dicarboxy-1,2,3,4,5, 8-hexamethylheme, a compact synthetic heme with the shortest acid side chains, to pursue the structural and functional consequences after intensive disruption of the heme propionate-apoglobin linkages in the native protein. The electron-withdrawing carboxylate groups directly attached to the porphyrin ring lowered the oxygen affinity by 3-fold as compared with native myoglobin. Autoxidation of the oxy derivative to the ferric protein proceeded with 1.6 x 10(-)2 min-1 at pH 7.0 and 30 degrees C. The crystallographic structure of the cyanomet myoglobin with 1.9 A resolution shows that the heme adopts a unique orientation in the protein pocket to extend the two carboxylates toward solvent sphere. The native globin fold is conserved, and the conformations of globin side chains are almost intact except for those located nearby the heme 6,7-carboxylates. The 7-carboxylate only weakly interacts with Ser92 and His97 through two mediating water molecules. The 6-carboxylate, on the other hand, forms a novel salt bridge with Arg45 owing to conformational flexibility of the guanidinium side chain. The proton NMR shows that the small heme does not fluctuate about the iron-histidine bond even at 55 degreesC, suggesting that the salt bridge between Arg45 and heme 6-carboxylate is of critical importance to recognize and fix the heme in myoglobin.

Of the 82 three dimensionally characterized residues of cytochrome c551, 49 are found to be structurally and topologically equivalent to the globin fold and 41 are equivalent to the cytochrome b5 fold, with a respective root mean square separation of 3.5 and 4.9 A between equivalenced Calpha atoms. The common fold represents a central heme binding core, corresponding to the middle exon of certain globin genes. After superposition of the protein folds, the heme irons are found to be separated by 5.4 and 1.6 A, while their heme normals are inclined by 6 degrees and 32 degrees, respectively. Furthermore, the heme "face", determined by the asymmetric attachment of the vinyl and propionyl side chains, is directed similarly in all three heme proteins. The heme itself is rotated by 72 degrees and 116 degrees about its normal, respectively. The minimum base change per codon for the three pairwise comparisons corresponds to the expected value of random sequence comparisons. While all three heme proteins may have diverged from a common ancestor, their similarity may have arisen from the requirements of heme binding or the utilization of a particularly stable fold. Known structures within commonly accepted divergent families were superimposed in order to discriminate better between convergence and divergence. Minimum base changes per codon, number of deletions and insertions, percentage of equivalenced residues, precision of heme superposition, and root mean square separation of equivalenced Calpha atoms were tested as measures of evolutionary relationships.