Hemin Loss Research Articles

Controlling hemoglobin-mediated lipid oxidation in herring (Clupea harengus) co-products via incubation or dipping in a recyclable antioxidant solution

Applying value-adding techniques to fish co-products is rendered difficult due to their high susceptibility to hemoglobin (Hb)-mediated lipid oxidation. In this study, we investigated a dipping technology with a solution containing Duralox MANC 213- a mixture of rosemary extract, ascorbic acid, tocopherols and citric acid – to control lipid oxidation during storage at 0 °C and 20 °C. The possibilities to re-use the antioxidant solution was also analyzed, along with studies on the link between Duralox MANC and Hb-form. Dipping in Duralox MANC largely increased the oxidation lag phase; from <0.5 to >3.5 d at 20 °C, and from <1 d to >11 d at 0 °C. Even after re-use of the solution up to 10 times, lipid oxidation was completely inhibited at 0 °C. Duralox MANC could prevent auto-oxidation and hemin loss of herring Hb; which are suggested as the main mechanisms behind the observed stabilization of herring co-products against lipid oxidation.

Identification of frozen/thawed beef based on label-free detection of hemin (Iron Porphyrin) with solution-gated graphene transistor sensors

The fraud labeling of frozen/thawed meat as fresh meat has become one of the severe problems in the current meat products market. Hemin, as a highly bioavailable iron form in beef, loses with exudates during freezing and thawing cycles. Based on this fact, in this work, solution-gated graphene transistor (SGGT) devices were constructed for discrimination of frozen/thawed beef. The detection mechanism of the sensor is mainly attributed to the adsorption of hemin molecules on the surface of graphene through ϕ-ϕ interaction, which leads to the change of doping level of graphene. The SGGT sensor for hemin shows a very low detection limit of 10 nM and a linear range of 100 nM 10 μM. Finally, determination of the number of freezing and thawing cycles by the rate of hemin loss detected by SGGT sensors for actual beef samples was successfully demonstrated.

Current Challenges in the Development of Acellular Hemoglobin Oxygen Carriers by Protein Engineering.

This article reviews the key biochemical mechanisms that govern O2 transport, NO scavenging, and oxidative degradation of acellular hemoglobin (Hb) and how these ideas have been used to try to develop strategies to engineer safer and more effective hemoglobin-based oxygen carriers (HBOCs). Significant toxicities due to acellular Hb have been observed after the administration of HBOCs or after the lysis of red cells, and include rapid clearance and kidney damage due to dissociation into dimers, haptoglobin binding, and macrophage activation; early O2 release leading to decreased tissue perfusion in capillary beds; interference with endothelial and smooth muscle signaling due to nitric oxide (NO) scavenging; autooxidization of heme iron followed by production of reactive oxygen species; and iron overload symptoms due to hemin loss, globin denaturation, iron accumulation, and further inflammation. Protein engineering can be used to mitigate some of these side effects, but requires an in-depth mechanistic understanding of the biochemical and biophysical features of Hb that regulate quaternary structure, O2 affinity, NO dioxygenation, and resistance to oxidation, hemin loss, and unfolding.

Engineering oxidative stability in human hemoglobin based on the Hb providence (βK82D) mutation and genetic cross-linking.

Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by H2O2 We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv βK82D mutation confers more resistance to degradation by H2O2, by markedly inhibiting oxidation of the β93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the βK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.

Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron 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 patient's 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.

Factors Governing Autooxidation of Human Hemoglobin

Determining mechanisms for the autooxidation of hemoglobin is required for understanding and treating unstable hemoglobinopathies and for developing more stable hemoglobin based O2 carriers. Previous studies suggested significant differences in autooxidation rates of α and β subunits. We used an azide reaction assay to measure the concentrations of ferric α and β chains at different time points during autooxidation. Our results showed no differences between the subunits. To obtain more accurate time courses for autooxidation, we deconvoluted observed spectra into the decay of HbO2, metHb appearance, hemichrome generation, and increases in turbidity due to hemin loss and apoprotein precipitation. The time courses for HbO2 decay at high concentrations (≥ 100µM heme) accelerate implying cooperative autooxidation, where as at low concentrations (≤ 10uM) the time courses are biphasic. These results suggest that the biphasic time courses at low hemoglobin are due to differences between tetramers and dimers. We have also measured autooxidation rates for a recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-α subunit. This hemoglobin shows a monophasic time course for autooxidation at both high and low protein concentrations, and the azide binding assay showed equal amounts of ferric α and β subunits. We have also examined recombinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed that the rate of autooxidation shows a bell-shaped dependence on oxygen concentration and increases markedly as the pH is decreased.Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert A. Welch Foundation.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

α-Hemoglobin Stabilizing Protein (AHSP) Markedly Decreases the Redox Potential and Reactivity of α-Subunits of Human HbA with Hydrogen Peroxide

α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobin A (HbA) and modulates heme iron oxidation and subunit folding states. Although AHSP·αHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. Both metHbA and isolated met-β-subunits undergo further oxidation in the presence of hydrogen peroxide (H(2)O(2)) to form ferryl heme species. Surprisingly, much lower levels of H(2)O(2)-induced ferryl heme are produced by free met-α-subunits as compared with met-β-subunits, and no ferryl heme is detected in H(2)O(2)-treated AHSP·met-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSP·met-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPR data suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and β-subunits. In contrast, treatment of free α-subunits with H(2)O(2) yields much smaller radical signals, and no radicals are detected when H(2)O(2) is added to AHSP·α-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from +40 to -78 mV in 1 m glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSP·met-α complex markedly decreases the rate of the initial H(2)O(2) reaction with iron and thus provides α-subunits protection against damaging oxidative reactions.

Development of recombinant hemoglobin-based oxygen carriers.

The worldwide blood shortage has generated a significant demand for alternatives to whole blood and packed red blood cells for use in transfusion therapy. One such alternative involves the use of acellular recombinant hemoglobin (Hb) as an oxygen carrier. Large amounts of recombinant human Hb can be expressed and purified from transgenic Escherichia coli. The physiological suitability of this material can be enhanced using protein-engineering strategies to address specific efficacy and toxicity issues. Mutagenesis of Hb can (i) adjust dioxygen affinity over a 100-fold range, (ii) reduce nitric oxide (NO) scavenging over 30-fold without compromising dioxygen binding, (iii) slow the rate of autooxidation, (iv) slow the rate of hemin loss, (v) impede subunit dissociation, and (vi) diminish irreversible subunit denaturation. Recombinant Hb production is potentially unlimited and readily subjected to current good manufacturing practices, but may be restricted by cost. Acellular Hb-based O(2) carriers have superior shelf-life compared to red blood cells, are universally compatible, and provide an alternative for patients for whom no other alternative blood products are available or acceptable. Remaining objectives include increasing Hb stability, mitigating iron-catalyzed and iron-centered oxidative reactivity, lowering the rate of hemin loss, and lowering the costs of expression and purification. Although many mutations and chemical modifications have been proposed to address these issues, the precise ensemble of mutations has not yet been identified. Future studies are aimed at selecting various combinations of mutations that can reduce NO scavenging, autooxidation, oxidative degradation, and denaturation without compromising O(2) delivery, and then investigating their suitability and safety in vivo.

Redox Instability and Hemin Loss of Mutant Sperm Whale Myoglobins Induced by 4-Hydroxynonenal in Vitro

The effects of 4-hydroxy-2-nonenal (HNE) on redox stability of Oxy- and Deoxy- wild-type (WT) and recombinant sperm whale myoglobins (P88H/Q152H, L29F, H97A, and H64F) and hemin loss from Met-myoglobin (Mb) were investigated. HNE induced greater redox instability in WT and mutant Mbs compared to controls (p < 0.05). The extent of HNE-induced OxyMb oxidation was lesser in L29F (p < 0.05) and greater in H97A and P88H/Q152H than in WT (p < 0.05). H64F DeoxyMb was more redox stable than WT DeoxyMb in the presence of HNE (p < 0.05). HNE alkylation occurred exclusively on histidine residues, and histidine 48 was alkylated in all sperm whale myoglobins. HNE alkylation accelerated the protoporphyrin moiety loss only in H97A. Met- forms of WT and L29F but not Deoxy- or Oxy- forms released hemin during storage. Primary structure strongly influenced Mb redox stability in the presence of reactive secondary lipid oxidation products.

Site E14 in Hemoglobins and Myoglobins: A Key Residue That Affects Hemin Loss and Lipid Oxidation Capacity

Fish hemoglobins (Hbs) frequently contain glycine at site E14 while mammalian Hbs contain larger residues (e.g., alanine and serine). These differences were examined by creating structural variants at E14 using recombinant bovine myoglobin (Mb) as a model heme protein that contains alanine at E14. The Ala(E14)Gly mutation increased k(ox) and hemin loss 3-fold and 45-fold, respectively. Glycine at E14 creates a channel for solvent to enter the heme crevice, which enhances autoxidation and hemin loss rates. Hydration of the proximal heme pocket facilitates hemin loss because protonation of the proximal histidine weakens the linkage of the imidazole group to the iron atom of the hemin moiety. Ala(E14)Gly promoted lipid oxidation in washed fish muscle more rapidly during iced storage compared to wild type Mb at pH 5.7. This suggested that the rapid hemin loss from Ala(E14)Gly accelerated lipid oxidation. Ala(E14)Ser and Ala(E14)Val had little effect on k(ox) but somewhat accelerated net hemin loss. These studies suggest that enhanced access of solvent to the heme crevice of many fish Hbs at site E14 facilitates rapid hemin loss and moderately accelerates autoxidation. This likely is part of the reason fish Hbs promote lipid oxidation much more effectively compared to mammalian Hbs.

The structure and NO binding properties of the nitrophorin-like heme-binding protein from Arabidopsis thaliana gene locus At1g79260.1.

The protein from Arabidopsis thaliana gene locus At1g79260.1 is comprised of 166-residues and is of previously unknown function. Initial structural studies by the Center for Eukaryotic Structural Genomics (CESG) suggested that this protein might bind heme, and consequently, the crystal structures of apo and heme-bound forms were solved to near atomic resolution of 1.32 A and 1.36 A, respectively. The rate of hemin loss from the protein was measured to be 3.6 x 10(-5) s(-1), demonstrating that it binds heme specifically and with high affinity. The protein forms a compact 10-stranded beta-barrel that is structurally similar to the lipocalins and fatty acid binding proteins (FABPs). One group of lipocalins, the nitrophorins (NP), are heme proteins involved in nitric oxide (NO) transport and show both sequence and structural similarity to the protein from At1g79260.1 and two human homologues, all of which contain a proximal histidine capable of coordinating a heme iron. Rapid-mixing and laser photolysis techniques were used to determine the rate constants for carbon monoxide (CO) binding to the ferrous form of the protein (k'(CO) = 0.23 microM(-1) s(-1), k(CO) = 0.050 s(-1)) and NO binding to the ferric form (k'(NO) = 1.2 microM(-1) s(-1), k(NO) = 73 s(-1)). Based on both structural and functional similarity to the nitrophorins, we have named the protein nitrobindin and hypothesized that it plays a role in NO transport. However, one of the two human homologs of nitrobindin contains a THAP domain, implying a possible role in apoptosis. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Effect of pH on Structural Changes in Perch Hemoglobin that Can Alter Redox Stability and Heme Affinity

pH can be manipulated to alter the oxidative stability of fish-based foods during storage. X-ray diffraction was used to investigate the ability of reduced pH to cause structural changes in fish hemoglobins that lead to enhanced oxidative degradation. Decreasing pH from 8.0 to 6.3 and 5.7 created a large channel for solvent entry into the heme crevice of perch hemoglobin beta chains. The proton-induced opening of this channel occurred between site CD3 and the heme-6-propionate. Solvent entry into the heme crevice can enhance metHb formation and hemin loss, processes that accelerate lipid oxidation. Reduced pH also decreased the distance between Ile at E11 in one of the alpha chains and the ligand above the heme iron atom. This sterically displaces O2 and protonated O2 which increases metHb formation. These studies demonstrate that pH reduction causes structural changes in perch hemoglobin which increase oxidative degradation of the heme pigment.

Involvement of Methemoglobin (MetHb) Formation and Hemin Loss in the Pro-oxidant Activity of Fish Hemoglobins

The capacity of Atlantic pollock ( Pollachius pollachius ), seabass ( Dicentrarchus labrax ), and horse mackerel ( Trachurus trachurus ) hemoglobin (Hb) to promote lipid oxidation has been evaluated in liposomes and washed minced horse mackerel muscle. The pro-oxidant ability of fish Hbs was related with their vulnerability to suffer oxidation to metHb and release hemin either in spontaneous situation or in the presence of two representative lipid oxidation products, linolein hydroperoxides and trans-2-pentenal. The results indicated similar effectiveness to promote lipid oxidation in liposomes and washed fish muscle: pollock Hb > horse mackerel Hb > seabass Hb. Pollock Hb showed a more elevated autoxidation rate and spontaneous hemin loss and also faster oxidation to metHb in the presence of hydroperoxides and trans-2-pentenal. The autoxidation and spontaneous hemin loss rates were intermediate for horse mackerel Hb, whereas seabass Hb exhibited the highest stability. The isoelectrofocusing (IEF) pattern of pollock Hb revealed the presence of isoforms with elevated anionic character, which are known to have poor oxygen affinity at the pH values found in fish muscle (pH 7.0-5.5). In agreement with the IEF patterns, pollock Hb was less oxygenated at pH 6.8, and seabass Hb exhibited more oxygenation than did horse mackerel. MetHb forms were significantly more effective in catalyzing lipid oxidation than the corresponding reduced fish Hbs and free hemin. The present investigation highlights a direct correlation between the pro-oxidant capacity of fish Hbs and their susceptibility to undergo metHb formation and hemin loss and also suggests a potential role of lipid oxidation byproducts in activating the pro-oxidative action of hemoglobin.

Structural analysis of fish versus mammalian hemoglobins: Effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.

Passively Released Heme from Hemoglobin and Myoglobin Is a Potential Source of Nutrient Iron for Bordetella bronchiseptica

Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.

Mechanisms of Heme Protein-Mediated Lipid Oxidation Using Hemoglobin and Myoglobin Variants in Raw and Heated Washed Muscle

The hemoglobin variant rHb 0.1, which possesses a decreased ability to form subunits, stimulated lipid oxidation in washed fish muscle less effectively as compared to wild-type hemoglobin (rHb 0.0). This could be due to the lower hemin affinity and more rapid autoxidation rate of subunits as compared to tetramers. To differentiate between hemin affinity and autoxidation effects, ferrous V68T Mb was compared to ferrous wild-type myoglobin (WT Mb). WT Mb has a more rapid hemin loss rate (25-fold) than does V68T, while V68T autoxidized more rapidly than did WT Mb (60-fold). Ferrous WT Mb promoted TBARS and lipid peroxide formation more rapidly than did ferrous V68T (p < 0.01). This indicated hemin loss rate was more critical in determining onset of lipid oxidation as compared to autoxidation rate. Hemin alone was capable of stimulating lipid oxidation. Albumin enhanced the ability of hemin to promote lipid oxidation. MetMb promoted lipid oxidation more effectively than did ferrous Mb, which could be due to the lower hemin affinity of metMb as compared to that of ferrous Mb. EDTA, an iron chelator, had no effect on the rate or extent of lipid oxidation mediated by Mb in the cooked system. Variants with a 975-fold range of hemin affinities promoted lipid oxidation with equivalent efficacy in cooked washed cod contrary to results in uncooked washed cod. The cooking temperatures apparently denature the globin and release hemin reactant to such an extent that the impact of hemin affinity on lipid oxidation observed in the raw state is negated in the cooked state. These studies collectively suggest released hemin is of primary importance in promoting lipid oxidation in raw and cooked washed fish muscle.

Probing the differences between rat liver outer mitochondrial membrane cytochrome b5 and microsomal cytochromes b5.

Two distinct forms of cytochrome b5 exist in the rat hepatocyte. One is associated with the membrane of the endoplasmic reticulum (microsomal, or Mc, cyt b5) while the other is associated with the outer membrane of liver mitochondria (OM cyt b5). Rat OM cyt b5, the only OM cyt b5 identified so far, has a significantly more negative reduction potential and is substantially more stable toward chemical and thermal denaturation than Mc cytochromes b5. In addition, hemin is kinetically trapped in rat OM cyt b5 but not in the Mc proteins. As a result, no transfer of hemin from rat OM cyt b5 to apomyoglobin is observed at pH values as low as 5.2, nor can the thermodyamically favored ratio of hemin orientational isomers be achieved under physiologically relevant conditions. These differences are striking given the similarity of the respective protein folds. A combined theoretical and experimental study has been conducted in order to probe the structural basis behind the remarkably different properties of rat OM and Mc cytochromes b5. Molecular dynamics (MD) simulations starting from the crystal structure of bovine Mc cyt b5 revealed a conformational change that exposes several internal residues to the aqueous environment. The new conformation is equivalent to the "cleft-opened" intermediate observed in a previously reported MD simulation of bovine Mc cyt b5 [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693]. The rat OM protein does not adopt a comparable conformation in MD simulations, thus restricting access of water to the protein interior. Subsequent comparisons of the protein sequences and structures suggested that an extended hydrophobic network encompassing the side chains of Ala-18, Ile-32, Leu-36, and Leu-47 might contribute to the inability of rat OM cyt b5 to adopt the cleft-opened conformation and, hence, stabilize its fold relative to the Mc isoforms. A corresponding network is not present in bovine Mc cyt b5 because positions 18, 32, and 47, are occupied by Ser, Leu, and Arg, respectively. To probe the roles played by Ala-18, Ile-32, and Leu-47 in endowing rat OM cyt b5 with its unusual structural properties, we have replaced them with the corresponding residues in bovine Mc cyt b5. Hence, the I32L (single), A18S/L47R (double), and A18S/L47R/I32L (triple) mutants of rat OM cyt b5 were prepared. The stability of these proteins was found to decrease in the following order: WT rat OM > rat OM I32L > rat OM A18S/L47R > rat OM A18S/L47R/I32L > bovine Mc cyt b5. The decrease in stability of the rat OM protein correlates with the extent to which the hydrophobic cluster involving the side chains of residues 18, 32, 36, and 47 has been disrupted. Complete disruption of the hydrophobic network in the triple mutant is confirmed in a 2.0 A resolution crystal structure of the protein. Disruption of the hydrophobic network also facilitates hemin loss at pH 5.2 for the double and triple mutants, with the less stable triple mutant exhibiting the greater rate of hemin transfer to apomyoglobin. Finally, 1H NMR spectroscopy and side-by-side comparisons of the crystal structures of bovine Mc, rat OM, and rat OM A18S/L47R/I32L cyt b5 allowed us to conclude that the nature of residue 32 plays a key role in controlling the relative stability of hemin orientational isomers A and B in rat OM cyt b5. A similar analysis led to the conclusion that Leu-70 and Ser-71 play a pivotal role in stabilizing isomer A relative to isomer B in Mc cytochromes b5.

Waterproofing the Heme Pocket: ROLE OF PROXIMAL AMINO ACID SIDE CHAINS IN PREVENTING HEMIN LOSS FROM MYOGLOBIN

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.