Heme Rotation Research Articles

Detecting rotational disorder in heme proteins: A comparison between resonance Raman spectroscopy, nuclear magnetic resonance, and circular dichroism

AbstractIn heme proteins, the canonical and reversed conformations result from the rotation of the heme group by 180° about the α,γ‐meso axis in the protein pocket. The coexistence of the two different heme orientations has been observed both in proteins reconstituted with hemin and in some native proteins. The reversal of the heme orientation can also change certain functional properties of heme proteins. Complementing the results from other experimental techniques, like circular dichroism and nuclear magnetic resonance, resonance Raman spectroscopy provides detailed information on the structure of the reversed heme. This allows one to elucidate the effects of the heme rotation on the vibrational spectra of the peripheral substituents, especially the vinyl groups. Furthermore, the combination of resonance Raman spectroscopy on single crystals and solution samples of heme proteins is proposed to be a sensitive tool to detect heme orientational disorder, even in the absence of structural data.

Assessment of Lymphoma and Other Hematologic Malignancies Training Needs Amongst Radiation Oncology Residents

The use of radiation therapy in the management of hematologic (heme) malignancies is heterogeneous and radiation oncology resident comfort with the management of this diverse group of diseases is unknown. Together, the International Lymphoma Radiation Oncology Group (ILROG) and Association of Residents in Radiation Oncology (ARRO) performed a study to assess current training opportunities for RO residents in treatment of heme patients and interest in a dedicated away elective to augment their existing training experience. RO residents (PGY2-5) in the ARRO email database (n=572) were sent an anonymous questionnaire in December 2018. Question types included binary, Likert-type scale (1=not at all, 5=extremely; reported as median [interquartile range]), and multiple choice. 134 residents (23%) completed the survey with majority of responses from PGY4/5 residents (64%) and in programs with ≥5 residents (93%). 112 residents (84%) reported having faculty who specialize in heme, and 95 residents (71%) reported having a heme rotation, most commonly ≥8 weeks in length. However, only 38 residents (40%) reported that these heme rotations were dedicated, i.e. >50% heme patients. Of 112 respondents, the percentage of residents reporting at least 4 cases with each diagnosis were: Non-Hodgkin lymphoma (83%), multiple myeloma (82%), cutaneous lymphoma (59%), Hodgkin lymphoma (53%), adult leukemia (46%). Residents reporting at least 4 cases of specialized treatment techniques were: breath hold for mediastinal lymphoma (39%), adult craniospinal irradiation (17%), total skin irradiation (39%), adult total body irradiation (68%), and proton therapy (36%). Residents reported feeling “moderately” prepared to advocate for radiation therapy in multidisciplinary conferences (3 [2-3]), to make clinical decisions once patients are referred (3 [2-4]), to oversee and critique treatment planning (3 [2-4]).They reported feeling “moderately” to “quite” prepared to contour heme cases using the involved site radiation therapy (3.5 [3-4]) and “quite” prepared to incorporate the Five Point Scale into treatment decisions (4 [3-5]). Overall, residents felt only “moderately” (3[2-3]) prepared to treat heme patients with 23% of residents responding that they felt “quite” or “extremely” comfortable. 49% of respondents were potentially interested in a RO away elective focused on heme, to increase comfort with treating heme patients (65%). Residents rated hands-on contouring modules, oral exams, and instruction on specialized techniques as the most worthwhile activities in an elective. Hematologic malignancies training is an important component of RO residency, yet less than 25% of surveyed trainees reported feeling well prepared to treat heme patients. Programs should explore opportunities to increase resident exposure; perhaps through participation in a dedicated heme elective in select programs or through on-line educational activities.

Utility of heme analogues to intentionally modify heme–globin interactions in myoglobin

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme–globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized.Proton NMR spectroscopy revealed that loose heme–globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron–histidine bond. The full rotational rate was estimated to be about 1400s−1 at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme–globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005mmHg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin.Heme–globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics – the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

3I0924 ヘムをヘム垂線のまわりに90°回転した時の共鳴ラマンスペクトル変化 : ヘムオキシゲネース(ヘム蛋白質,口頭発表)

3I0924 ヘムをヘム垂線のまわりに90°回転した時の共鳴ラマンスペクトル変化 : ヘムオキシゲネース(ヘム蛋白質,口頭発表)

Resonance Raman Interrogation of the Consequences of Heme Rotational Disorder in Myoglobin and Its Ligated Derivatives

Resonance Raman spectroscopy is employed to characterize heme site structural changes arising from conformational heterogeneity in deoxyMb and ligated derivatives, i.e., the ferrous CO (MbCO) and ferric cyanide (MbCN) complexes. The spectra for the reversed forms of these derivatives have been extracted from the spectra of reconstituted samples. Dramatic changes in the low-frequency spectra are observed, where newly observed RR modes of the reversed forms are assigned using protohemes that are selectively deuterated at the four methyl groups or at the four methine carbons. Interestingly, while substantial changes in the disposition of the peripheral vinyl and propionate groups can be inferred from the dramatic spectral shifts, the bonds to the internal histidyl imidazole ligand and those of the Fe-CO and Fe-CN fragments are not significantly affected by the heme rotation, as judged by lack of significant shifts in the nu(Fe-N(His)), nu(Fe-C), and nu(C-O) modes. In fact, the apparent lack of an effect on these key vibrational parameters of the Fe-N(His), Fe-CO, and Fe-CN fragments is entirely consistent with previously reported equilibrium and kinetic studies that document virtually identical functional properties for the native and reversed forms.

Effect of the N-Terminus on Heme Cavity Structure, Ligand Equilibrium, Rate Constants, and Reduction Potentials of Nitrophorin 2 from Rhodnius prolixus

The D1A mutant of recombinant NP2 has been prepared and shown to have the expression-initiation methionine-0 cleaved during expression in E. coli, as is the case for recombinant NP4, where Ala is the first amino acid for the recombinant protein as well as for the mature native protein. The heme substituent 1H NMR chemical shifts of NP2-D1A and those of its imidazole, N-methylimidazole, and cyanide complexes are rather different from those of NP2-M0D1. This difference is likely due to the much smaller size of the N-terminal amino acid (A) of NP2-D1A, which allows the formation of the closed loop form of this protein, as it does for NP4 (Weichsel, A., Andersen, J. F., Roberts, S. A., and Montfort, W. R. (2000) Nature Struct. Biol. 7, 551-554). The ratio of the two hemin rotational isomers A and B is different for the two proteins, and the rate at which the A:B ratio reaches equilibrium is strikingly different (NP2-M0D1 t1/2 for heme rotation approximately 2 h, NP2-D1A t1/2 approximately 43 h). This difference is consistent with the high stability of the closed loop form of the NP2-D1A protein and infrequent opening of the loops that could allow heme to at least partially exit the binding pocket in order to rotate about its alpha,gamma-meso axis. Consistent with this, the rates of histamine binding and release to/from NP2-D1A are significantly slower than those for NP2-M0D1 at pH 7.5. This work suggests that care must be taken in interpreting data obtained from proteins that carry the expression-initiation M0.

Proton NMR study of myoglobin reconstituted with 3,7-diethyl-2,8-dimethyl iron porphyrin: Remarkable influence of peripheral substitution on heme rotation

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N–H resonance at 82.5 ppm to indicate the formation of the Fe N (His93) bond. The pyrrole–protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between −10 and −18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole–protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.

Steric effects of isoleucine 107 on heme reorientation reaction in human myoglobin

Structural factors to regulate the heme reorientation reaction in myoglobin were examined and we found that the side chain at position 107 (Ile107), which is located between the 2-vinyl and 3-methyl groups of heme, forms a kinetic barrier for the heme rotation about the α– γ axis. The phenylalanine-substituted mutant showed an extremely slow heme reorientation rate, compared to that of the wild-type protein, while replacement by the decreased side chain, valine, at position 107 accelerated the reorientation reaction. Considering that the spectroscopic data show only minor structural changes in the heme environments of the Ile107 mutants, the side chain at position 107 sterically interacts with the heme peripheral groups in the activation state for the heme reorientation, which supports the intramolecular mechanism that the heme rotates about the α– γ axis without leaving the “protein cage.”

Effect of rapid heme rotation on electrochemistry of myoglobin

Myoglobins (Mbs) reconstituted with rotatable octamethylheme and non-rotatable etioheme were prepared and their electrochemical behavior was studied. The redox potential of octamethylheme reconstituted Mb (OMe–Mb), of which heme rotates around iron–histidine (F8His) bond, shifted negatively by ca. 30 mV compared with non-rotatable etioheme reconstituted Mb (Etio–Mb). On the other hand, the redox potentials of octamethylheme and etioheme themselves were very similar to each other. Due to the similarity of the distal histidine side of the heme of these two reconstituted Mbs, the shifts of the redox potential would be attributable to the drastic change of the orientation of proximal histidine imidazole ring to the heme plane by heme rotation. The dissociation rate constant of cyanide ion from the ferrous heme iron (II) for OMe–Mb form at 5°C and pH 7.5 was three times faster than that of Etio–Mb. The electron transfer kinetics of these Mbs showed that the heme rotation causes faster electron transfer rates in both electrode reaction and chemical reduction in solution with dithionite. The obtained heterogeneous electron transfer rates constants at an In 2O 3 electrode and first-order rate constants of the chemical reduction were 12(±0.5)×10 −4 cm s −1, 9.8(±1.0) s −1 for OMe–Mb and 6.0(±0.5)×10 −4 cm s −1, 4.5(±1.0) s −1 for Etio–Mb under the present experimental conditions.

1H-NMR investigation of the influence of the heme orientation on functional properties of myoglobin

There are two interconverting forms of myoglobin, which differ in the orientation of the heme by a 180° rotation around the α,γ-meso axis; the proteins possessing the same heme orientation, as found in the single crystal, and the reversed heme orientation are called the major and minor forms, respectively. Structures and functional properties of these two forms have been investigated by NMR. Heme peripheral side-chain and non-coordinated amino acid proton resonances of the minor form in its met-cyano form have been assigned and the comparison of the shift between the corresponding resonances of the two forms revealed that the heme electronic structure is not largely influenced by the heme rotation. On the other hand, the exchange rate of His E7 N ϵH proton of the minor form is larger by a factor of 3–5 than that of the major one, indicating that the stability of the hydrogen bond between Fe-bound ligand and His E7 is slightly weaker in the minor form that in the major one. The rate of autoxidation of oxy-myoglobin and azide affinity of met-aquo myoglobin were also found to depend on the orientation of the heme.

Consequence of rapid heme rotation to the oxygen binding of myoglobin

The myoglobin complexed with octamethylhemin was prepared. The visible absorption profiles are typical of general alkylhemin-substituted myoglobins, suggesting normal insertion of the hemin into the hydrophobic cavity. The NMR spectra of various ferric proteins were anomalous, without clearly resolved signals from the prosthetic group, most probably reflecting rapid heme rotation about the iron-histidine bond. The equilibrium and kinetic oxygen bindings were measured to examine the functional significance of the heme motion. Functional comparison with the myoglobin having immobile hemin indicates that rotation of the octamethylheme negligibly affects the myoglobin function. The results suggest that neither the heme peripheral contacts nor the proximal imidazole orientation about the heme normal is the dominant factor to control myoglobin function.

Structural analysis of the myoglobin reconstituted with iron porphine.

Sperm whale apomyoglobin was complexed with iron porphine to examine the influence of completely removed heme side chains on the entire molecular structure. Paramagnetic NMR peak from the proximal histidine of the deoxy protein ensured formation of the iron-histidine bond. Porphine pyrrole-proton NMR signals of the cyanmet and deoxy derivatives are unusually sharp single lines manifesting rapid heme rotation about the iron-histidine bond. X-ray crystallographic structure of the cyanmet derivative, determined with a final R factor of 0.21 for 11,808 independent reflections ranging from 7 to 1.8 A, was resolved at 1.8 A resolution. The result confirmed 1:1 coupling between apomyoglobin and iron porphine. The cyano ligand adopts a bent configuration with an Fe-C-N angle of 127 degrees and a Fe-CN distance of 1.89 A. The overall globin structure and side chain conformations are remarkably similar to those of native myoglobin despite intensive disruption of the original heme-globin interactions. The native apoprotein structure unexpectedly conserved even after iron porphine insertion demonstrates that the complex polypeptide fold of holomyoglobin is more inherent in the amino acid sequence than is generally believed.

Dynamic analysis of efficient heme rotation in myoglobin by NMR spectroscopy

Sperm whale myoglobin was reconstituted with 1,4,5,8-tetramethylhemin. The hyperfine-shifted proton NMR signals from the prosthetic group exhibit remarkable patten changes around 15°C, while the globin resonances are normal to obey the Curie law. The NMR anomaly specifically observed for the heme signals suggests a slow to rapid rotational transition of the hemin about the iron-histidine bond. The temperature-dependent pattern changes were quantitatively analyzed by a dynamic NMR method. Two sets of analyses with the heme-methyl and pyrrole-proton lines consistently afforded ΔH ≠ = 16.3 kcal/mol, ΔS ≠ = 14.0 e.u, ΔG ≠ = 12.1 kcal/mol at 298 K, and a frequency of 90° heme rotation 5600 s −1 at 20°C. The relatively large activation entropy suggests that structural rearrangements at the direct heme vicinity are involved and that efficient heme rotation is accomplished by a number of fluctuative local heme-globin contacts within a conserved crevice structure.

Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: evidence for intramolecular reorientation about a heme two-fold axis

Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: evidence for intramolecular reorientation about a heme two-fold axis