Dioxygenation of human serum albumin having a prosthetic heme group in a tailor-made heme pocket

Human serum albumin (HSA) is the most abundant plasma protein in our bloodstream and serves as a transporter for small hydrophobic molecules such as fatty acids, bilirubin, and steroids. Hemin dissociated from methemoglobin is also bound within a narrow D-shaped cavity in subdomain IB of HSA. In terms of the general hydrophobicity of the alpha-helical pocket, HSA potentially has features similar to the heme-binding site of myoglobin (Mb) or hemoglobin (Hb). However, the reduced ferrous HSA-heme complex is immediately oxidized by O2, because HSA lacks the proximal histidine that enables the heme group to bind O2. In this paper, we report the introduction of a proximal histidine into the subdomain IB of HSA by site-directed mutagenesis to construct a tailor-made heme pocket (I142H/Y161L), which allows a reversible O2 binding to the prosthetic heme group. Laser flash photolysis experiments revealed that this artificial hemoprotein appears to have two different geometries of the axial-imidazole coordination, and these two species (I and II) showed rather low O2 binding affinities (P1/2O2 = 18 and 134 Torr) relative to those of Mb and Hb.     

Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti

Background: The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity. On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase. This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy.Results: Using one-dimensional N MR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively. We determined the hyperfine-shifted ¹H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals.Conclusions: These findings support the spin-state mechanism of FixL regulation. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.     

Carbon monoxide binding by de novo heme proteins derived from designed combinatorial libraries.

Carbon monoxide binding was studied in a collection of de novo heme proteins derived from combinatorial libraries of sequences designed to fold into 4-helix bundles. The design of the de novo sequences was based on the previously reported "binary code" strategy, in which the patterning of polar and nonpolar amino acids is specified explicitly, but the exact identities of the side chains are varied extensively.(1) The combinatorial mixture of amino acids included histidine and methionine, which ligate heme iron in natural proteins. However, no attempt was made to explicitly design a heme binding site. Nonetheless, as reported previously, approximately half of the binary code proteins bind heme.(2) This collection of novel heme proteins provides a unique opportunity for an unbiased assessment of the functional potentialities of heme proteins that have not been prejudiced either by explicit design or by evolutionary selection. To assess the capabilities of the de novo heme proteins to bind diatomic ligands, we measured the affinity for CO, the kinetics of CO binding and release, and the resonance Raman spectra of the CO complexes for eight de novo heme proteins from two combinatorial libraries. The CO binding affinities for all eight proteins were similar to that of myoglobin, with dissociation constants (K(d)) in the low nanomolar range. The CO association kinetics (k(on)) revealed that the heme environment in all eight of the de novo proteins is partially buried, and the resonance Raman studies indicated that the local environment around the bound CO is devoid of hydrogen-bonding groups. Overall, the CO binding properties of the de novo heme proteins span a narrow range of values near the center of the range observed for diverse families of natural heme proteins. The measured properties of the de novo heme proteins can be considered as a "default" range for CO binding in alpha-helical proteins that have neither been designed to bind heme or CO, nor subjected to genetic selections for heme or CO binding.     

Characterization of Mononuclear Non‐heme Iron(III)‐Superoxo Complex with a Five‐Azole Ligand Set

Reaction of O₂ with a high‐spin mononuclear iron(II) complex supported by a five‐azole donor set yields the corresponding mononuclear non‐heme iron(III)–superoxo species, which was characterized by UV/Vis spectroscopy and resonance Raman spectroscopy. ¹H NMR analysis reveals diamagnetic nature of the superoxo complex arising from antiferromagnetic coupling between the spins on the low‐spin iron(III) and superoxide. This superoxo species reacts with H‐atom donating reagents to give a low‐spin iron(III)–hydroperoxo species showing characteristic UV/Vis, resonance Raman, and EPR spectra.     

Photodissociation of heme distal methionine in ferrous cytochrome C revealed by subpicosecond time-resolved resonance Raman spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.     

布洛芬与人血白蛋白位点II动态结合过程的分子模拟:一种结合途径分析

人血白蛋白（HSA）主要有两个药物结合位点,位点I和位点Ⅱ,许多小分子优先结合在位点Ⅱ上,包括抗炎类药物布洛芬。本文采用分子模拟方法研究了布洛芬小分子与HSA位点Ⅱ结合的动态过程,探讨了二者的结合机制。首先构建了50个随机分布的布洛芬与HSA复合物体系,经50 ns分子动力学模拟,其中一个布洛芬分子稳定结合于位点Ⅱ。基于该分子的运动轨迹分析,发现布洛芬的结合可分为四个阶段,即远程吸引、表面结合调整、进入位点Ⅱ空腔和稳定结合。比较范德华和静电相互作用能,发现初期以静电吸引为主,中期在HSA表面的两个极性区域间调整,逐步转移至位点Ⅱ附近;然后在位点Ⅱ入口处的极性残基和附近疏水残基的共同作用下,布洛芬进入位点Ⅱ空腔;进入空腔后,静电和疏水共同作用形成稳定结合。在结合过程中,位点Ⅱ附近的蛋白表面发生明显改变,体现出一定的“诱导契合”作用,同时分子模拟得到的结合模式和布洛芬-HSA结合的晶体结构类似。结果表明,分子模拟可以辅助研究小分子和蛋白结合的动态过程,从分子水平阐述相关结合机制。     

Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

Iron twin-coronet porphyrins as models of myoglobin and hemoglobin: amphibious electrostatic effects of overhanging hydroxyl groups for successful CO/O2 discrimination

Inspired by the observation of polar interactions between CO and O(2) ligands and the peptide residues at the active site of hemoglobin and myoglobin, we synthesized two kinds of superstructured porphyrins: TCP-IM, which contains a linked imidazole ligand, and TCP-PY, which contains a linked pyridine ligand, and examined the thermodynamic, kinetic, and spectroscopic (UV/Vis, IR, NMR, and resonance Raman) properties of their CO and O(2) complexes. On both sides of each porphyrin plane, bulky binaphthyl bridges form hydrophobic cavities that are suitable for the binding of small molecules. In the proximal site, an imidazole or pyridine residue is covalently fixed and coordinates axially to the central iron atom. In the distal site, two naphtholic hydroxyl groups overhang toward the center above the heme. The CO affinities of TCPs are significantly lower than those of other heme models. In contrast, TCPs have moderate O(2) binding ability. Compared with reported model hemes, the binding selectivity of O(2) over CO in TCP-IM and TCP-PY complexes is greatly improved. The high O(2) selectivity of the TCPs is mainly attributable to a low CO affinity. The comparison of k(on)(CO) values of TCPs with those of unhindered hemes indicates the absence of steric hindrance to the intrinsically linear CO coordination to Fe(II) in TCP-IM and TCP-PY. The abnormally large k(off)(CO) values are responsible for the low CO affinities. In contrast, k(off)(O(2)) of TCP-PY is smaller than those of other pyridine-coordinated model hemes. For the CO adducts of TCPs, unusually low nu(Fe-CO) and unusually high nu(C-O) frequencies are observed. These results can be ascribed to decreased back-bonding from the iron atom to the bound CO. The lone pairs of the oxygen atoms of the hydroxyl groups prevent back-bonding by exertion of a strong negative electrostatic interaction. On the other hand, high nu(Fe-O(2)) frequencies are observed for the O(2) adducts of TCPs. In the resonance Raman (RR) spectrum of oxy-TCP-IM, we observed simultaneous enhancement of the Fe-O(2) and O-O stretching modes. Furthermore, direct evidence for hydrogen bonding between the hydroxyl groups and bound dioxygen was obtained by RR and IR spectroscopy. These spectroscopic data strongly suggest that O(2) and CO binding to TCPs is controlled mainly by the two different electrostatic effects exerted by the overhanging OH groups: destabilization of CO binding by decreasing back-bonding and stabilization of O(2) binding by hydrogen bonding.     

Insight into heme protein redox potential control and functional aspects of six-coordinate ligand-sensing heme proteins from studies of synthetic heme peptides

We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2III > K1III; K2II < K1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.     

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.     

Characterizing Active Pharmaceutical Ingredient Binding to Human Serum Albumin by Spin‐Labeling and EPR Spectroscopy

Drug binding to human serum albumin (HSA) has been characterized by a spin‐labeling and continuous‐wave (CW) EPR spectroscopic approach. Specifically, the contribution of functional groups (FGs) in a compound on its albumin‐binding capabilities is quantitatively described. Molecules from different drug classes are labeled with EPR‐active nitroxide radicals (spin‐labeled pharmaceuticals (SLPs)) and in a screening approach CW‐EPR spectroscopy is used to investigate HSA binding under physiological conditions and at varying ratios of SLP to protein. Spectral simulations of the CW‐EPR spectra allow extraction of association constants (K_A) and the maximum number (n) of binding sites per protein. By comparison of data from 23 SLPs, the mechanisms of drug–protein association and the impact of chemical modifications at individual positions on drug uptake can be rationalized. Furthermore, new drug modifications with predictable protein binding tendency may be envisaged.     

Probing the environment of cu(b) in heme-copper oxidases

Time-resolved step-scan FTIR (TRS2-FTIR) and density functional theory have been applied to probe the structural dynamics of CuB in heme-copper oxidases at room temperature. The TRS2-FTIR data of cbb3 from Pseudomonas stutzeri indicate a small variation in the frequency of the transient CO bound to CuB in the pH/pD 7-9 range. This observation in conjunction with density functional theory calculations, in which significant frequency shifts of the nu(CO) are observed upon deprotonation and/or detachment of the CuB ligands, demonstrates that the properties of the CuB ligands including the cross-linked tyrosine, in contrast to previous reports, remain unchanged in the pH 7-9 range. We attribute the small variations in the nu(CO) of CuB to protein conformational changes in the vicinity of CuB. Consequently, the split of the heme Fe-CO vibrations (alpha-, beta-, and gamma-forms) is not due to changes in the ligation and/or protonation states of the CuB ligands or to the presence of one or more ionizable groups, as previously suggested, but the result of global protein conformational changes in the vicinity of CuB which, in turn, affect the position of CuB with respect to the heme Fe.     

Binding of YC-1 or BAY 41-2272 to soluble guanylyl cyclase induces a geminate phase in CO photolysis

Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of approximately 150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 x 10(7) s(-1). These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.     

Weak-field anions displace the histidine ligand in a synthetic heme peptide but not in N-acetylmicroperoxidase-8: possible role of heme geometry differences

We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.     

Spin Crossover in Nitrito‐Myoglobin as Revealed by Resonance Raman Spectroscopy

The myoglobin (Mb) heme Fe‐O‐N=O and heme Fe‐O‐N=O/2‐nitrovinyl species have been characterized by resonance Raman spectroscopy. In the heme Fe‐O‐N=O species, the bound nitrite ligand is removed by solvent exchange, thus reforming metmyoglobin (metMb). The high‐spin heme Fe‐O‐N=O unit is converted into a low‐spin heme Fe‐O‐N=O/2‐nitrovinyl species that can be reversibly switched between a low‐ and a high‐spin state without removing the bound nitrite ligand, as observed in the case of the heme Fe‐O‐N=O species. This spin‐state change is likely to be accompanied by a general structural rearrangement in the protein‐binding pocket. This example is the first of a globin protein that can reversibly change its metal spin state through an internal perturbation. These findings provide a basis for understanding the structure–function relationship of the spin cross found in other metalloenzymes and Fe^III–porphyrin complexes.     

Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.     

A density functional theory investigation of Fe-N-O bonding in heme proteins and model systems

We report the results of a series of density functional theory (DFT) calculations of the M?ssbauer quadrupole splittings and isomer shifts in NO heme model compounds, together with the results of calculations of the M?ssbauer quadrupole splittings, isomer shifts, and electron paramagnetic resonance hyperfine coupling constants in a model Fe(II)(NO)(imidazole) complex as a function of Fe-NO bond length and Fe-N-O bond angle. The results of the M?ssbauer quadrupole splitting and isomer shift calculations on the NO heme model compounds show good accord between theory and experiment, with the largest errors being observed for structures having the largest crystallographic R(1) values. The results of the property surface calculations were then used to calculate Fe-NO bond length and Fe-N-O bond angle probability surfaces (Z-surfaces) for a nitrosyl hemoglobin, using, in addition, an energy filter. The results obtained yielded a most probable Fe-NO bond length (r) of 1.79 A and an Fe-N-O bond angle (beta) of 136 degrees -137 degrees. This bond length is somewhat longer than those observed in most model compounds but may be due, at least in part, to hydrogen bond formation with the distal His residue. Bond elongation was also observed in a geometry optimized Fe(II)(NO)(imidazole) complex hydrogen bonded to an imidazole residue, in which we find r = 1.76-1.78 A and beta = 137 degrees -138 degrees. The computed bond angles are close to the canonical approximately 140 degrees value found in most model systems. Highly bent Fe-N-O bond angles or very long Fe-NO bond lengths seem unlikely to occur in proteins, due to their high energies. We also investigated the molecular orbitals and spin densities in each of the six coordinate systems investigated and found the orbitals and spin densities to be generally similar those described previously for five coordinate systems. Taken together, these results show that M?ssbauer quadrupole splittings and isomer shifts, in addition to electron paramagnetic resonance hyperfine coupling constants, can now be calculated for nitrosyl heme systems with relatively good accuracy and that the results so obtained can be used to determine Fe-N-O geometries in metalloproteins. The Z-surface approach is thus applicable to both diamagnetic (CO) and paramagnetic (NO) heme proteins with in both cases the metal-ligand binding geometries found in the proteins being very close to those seen in model systems.     

Theoretical density functional theory studies on interactions of small biologically active molecules with isolated heme group

We present ab-initio density functional theory studies on the interactions of small biologically active molecules, namely NO, CO, O(2), H(2)O, and NO(2) (-) with the full-size heme group. Our results show that the small molecule-iron bond is the strongest in carbonyl and the weakest in nitrite system. Trans influence induced by NO binding to the five-coordinate heme complex is shown. Nitric oxide in the resulting complex might be described as NO(-). The differences among the small ligands of XO type (CO, NO, O(2)), and their distant chemical behavior from H(2)O and NO(2) (-) ligands in binding to the Fe(II) ion, are shown. Moreover, the role of the heme ring as a reservoir of electrons in the studied complexes is invoked. The analysis of the parameters defining the iron-histidine bond indicates that this bond is longer and weaker in nitrosyl and carbonyl complexes than in the other systems. Our findings support the proposed mechanism of soluble guanylate cyclase (sGC) activation and suggest that the first step of sGC activation by CO may be the same as during the activation by NO. Obtained results are then compared with the data concerning smaller model of the heme, the porphyrin complexes, available in the literature.     

鼠脑红蛋白突变体F106L与氰根的结合作用

构建了鼠脑红蛋白(Mouse neuroglobin)的突变体F106L, 以探求近端残基对脑红蛋白血红素口袋结构的贡献. 通过溶液核磁共振方法研究了外来配体氰根离子与NgbF106L蛋白的结合作用, 结果显示, 此结合存在动力学过程, 并且NgbF106LCN 突变蛋白氰根络合物可以可逆地释放氰根离子, 并使原来的第6配体His64(E7)又结合回到血红素铁上. 研究结果揭示, G5(Phe106)残基对脑红蛋白血红素构象而言较为保守; QM/MM结构优化结果表明, 位于G5 和FG5的近端残基对蛋白结构稳定性具有重要作用, 并可调控外来配体与蛋白作用的配位平衡与热动力学性质.