Heterogeneous kinetics of the carbon monoxide association and dissociation reaction to nitrophorin 4 and 7 coincide with structural heterogeneity of the gate-loop.

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme-CO complexes of NP4, NP4(D30N), and NP7 as isoelectronic models for the ferriheme-NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-IR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leu130 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs.

[1]  M. H. Pereira,et al.  Identification of the native N-terminus of the membrane attaching ferriheme protein nitrophorin 7 from Rhodnius prolixus. , 2012, Analytical biochemistry.

[2]  Chunmao He,et al.  Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction. , 2011, Journal of inorganic biochemistry.

[3]  S. Neya,et al.  Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins. , 2011, Biochemistry.

[4]  H. Ogata,et al.  P48. Nitrite disproportionation reaction: Investigations on the mechanism of the conversion of nitrite into nitric oxide at the ferriheme center of nitrophorins at blood plasma pH , 2011 .

[5]  Chunmao He,et al.  Nitrophorins: Nitrite disproportionation reaction and other novel functionalities of insect heme‐based nitric oxide transport proteins , 2011, IUBMB life.

[6]  H. Ogata,et al.  Formation of the complex of nitrite with the ferriheme b beta-barrel proteins nitrophorin 4 and nitrophorin 7. , 2010, Biochemistry.

[7]  M. Kubo,et al.  Ultrafast dynamics of diatomic ligand binding to nitrophorin 4. , 2010, Journal of the American Chemical Society.

[8]  G. Chaudhuri,et al.  NO to breast: when, why and why not? , 2010, Current pharmaceutical design.

[9]  F. Spyrakis,et al.  Ligand migration through the internal hydrophobic cavities in human neuroglobin , 2009, Proceedings of the National Academy of Sciences.

[10]  Chunmao He,et al.  Formation of nitric oxide from nitrite by the ferriheme b protein nitrophorin 7. , 2009, Journal of the American Chemical Society.

[11]  M. Shokhirev,et al.  Effect of mutation of carboxyl side-chain amino acids near the heme on the midpoint potentials and ligand binding constants of nitrophorin 2 and its NO, histamine, and imidazole complexes. , 2009, Journal of the American Chemical Society.

[12]  A. Roitberg,et al.  pH-dependent mechanism of nitric oxide release in nitrophorins 2 and 4. , 2009, The journal of physical chemistry. B.

[13]  E. Wagner,et al.  Nitric oxide--a novel therapeutic for cancer. , 2008, Nitric oxide : biology and chemistry.

[14]  M. Ibrahim,et al.  DFT analysis of axial and equatorial effects on heme-CO vibrational modes: applications to CooA and H-NOX heme sensor proteins. , 2008, Biochemistry.

[15]  Alessandra Pesce,et al.  Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem‐reactivity , 2008, EMBO reports.

[16]  M. Shokhirev,et al.  Spectroscopic and functional characterization of nitrophorin 7 from the blood-feeding insect Rhodnius prolixus reveals an important role of its isoform-specific N-terminus for proper protein function. , 2007, Biochemistry.

[17]  F. Spyrakis,et al.  Ligand migration in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana. , 2007, The journal of physical chemistry. B.

[18]  F. Walker,et al.  Overexpression in Escherichia coli and functional reconstitution of the liposome binding ferriheme protein nitrophorin 7 from the bloodsucking bug Rhodnius prolixus. , 2007, Protein expression and purification.

[19]  Zhi Huang,et al.  Nitric oxide red blood cell membrane permeability at high and low oxygen tension. , 2007, Nitric oxide : biology and chemistry.

[20]  F. Spyrakis,et al.  The reactivity with CO of AHb1 and AHb2 from Arabidopsis thaliana is controlled by the distal HisE7 and internal hydrophobic cavities. , 2007, Journal of the American Chemical Society.

[21]  A. Mozzarelli,et al.  Time-resolved methods in Biophysics. 2. Monitoring haem proteins at work with nanosecond laser flash photolysis , 2006, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[22]  Q. Qi,et al.  A Glycosylated Nitric Oxide Donor, β‐Gal‐NONOate, and its Site‐specific Antitumor Activity , 2006 .

[23]  M. Knipp.  How to Control NO Production in Cells: Nω,Nω‐Dimethyl‐L‐Arginine Dimethylaminohydrolase as a Novel Drug Target , 2006 .

[24]  M. Teixeira,et al.  Salivation pattern of Rhodnius prolixus (Reduviidae; Triatominae) in mouse skin. , 2006, Journal of insect physiology.

[25]  A. Mozzarelli,et al.  Determination of microscopic rate constants for CO binding and migration in myoglobin encapsulated in silica gels. , 2005, The journal of physical chemistry. B.

[26]  B. Noll,et al.  Heme carbonyls: environmental effects on nu(C-O) and Fe-C/C-O bond length correlations. , 2005, Journal of the American Chemical Society.

[27]  W. Montfort,et al.  Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes. , 2005, Biochemistry.

[28]  J. Andersen,et al.  Structural Determinants of Factor IX(a) Binding in Nitrophorin 2, a Lipocalin Inhibitor of the Intrinsic Coagulation Pathway* , 2005, Journal of Biological Chemistry.

[29]  C. Viappiani,et al.  Kinetics of proton release after flash photolysis of 1-(2-nitrophenyl)ethyl sulfate (caged sulfate) in aqueous solution. , 2005, Journal of the American Chemical Society.

[30]  J. Andersen,et al.  The role of salivary lipocalins in blood feeding by Rhodnius prolixus. , 2005, Archives of insect biochemistry and physiology.

[31]  Walker Fa.  Nitric oxide interaction with insect nitrophorins and thoughts on the electron configuration of the {FeNO}6 complex. , 2005 .

[32]  T. Spiro,et al.  CO as a vibrational probe of heme protein active sites. , 2005, Journal of inorganic biochemistry.

[33]  V. Balakotaiah,et al.  Diffusing capacity reexamined: relative roles of diffusion and chemical reaction in red cell uptake of O2, CO, CO2, and NO. , 2004, Journal of applied physiology.

[34]  D. Kondrashov,et al.  Protein functional cycle viewed at atomic resolution: conformational change and mobility in nitrophorin 4 as a function of pH and NO binding. , 2004, Biochemistry.

[35]  G. Nienhaus,et al.  Structural Dynamics Controls Nitric Oxide Affinity in Nitrophorin 4* , 2004, Journal of Biological Chemistry.

[36]  J. Andersen,et al.  Recognition of anionic phospholipid membranes by an antihemostatic protein from a blood-feeding insect. , 2004, Biochemistry.

[37]  J. Andersen,et al.  Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4. , 2004, Biochemistry.

[38]  A. Mozzarelli,et al.  CO rebinding kinetics to myoglobin- and R-state-hemoglobin-doped silica gels in the presence of glycerol , 2004 .

[39]  F. Walker,et al.  Electrochemical and NMR spectroscopic studies of distal pocket mutants of nitrophorin 2: Stability, structure, and dynamics of axial ligand complexes , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[40]  E. Henry,et al.  A tertiary two-state allosteric model for hemoglobin. , 2002, Biophysical chemistry.

[41]  F. Walker,et al.  Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4. , 2001, Biochemistry.

[42]  Jean G. Riess,et al.  Oxygen Carriers (“Blood Substitutes”)Raison d'Etre, Chemistry, and Some Physiology Blut ist ein ganz besondrer Saft1 , 2001 .

[43]  N. Wengenack,et al.  Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding. , 2001, Biochemistry.

[44]  J. Andersen,et al.  The Crystal Structure of Nitrophorin 2 , 2000, The Journal of Biological Chemistry.

[45]  J. Andersen,et al.  Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap. , 2000, Biochemistry.

[46]  J. Andersen,et al.  Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial , 2000, Nature Structural Biology.

[47]  T. Spiro,et al.  Role of the axial ligand in hemeCO backbonding; DFT analysis of vibrational data , 2000 .

[48]  B. McMahon,et al.  Connection between the taxonomic substates and protonation of histidines 64 and 97 in carbonmonoxy myoglobin. , 1999, Biophysical journal.

[49]  J. Andersen,et al.  Nitric Oxide Binding to the Ferri- and Ferroheme States of Nitrophorin 1, a Reversible NO-Binding Heme Protein from the Saliva of the Blood-Sucking Insect, Rhodnius prolixus , 1999 .

[50]  J. Andersen,et al.  The crystal structure of nitrophorin 4 at 1.5 A resolution: transport of nitric oxide by a lipocalin-based heme protein. , 1998, Structure.

[51]  K. Shikama.  The Molecular Mechanism of Autoxidation for Myoglobin and Hemoglobin: A Venerable Puzzle. , 1998, Chemical reviews.

[52]  J. Olson,et al.  Disruption of the heme iron-proximal histidine bond requires unfolding of deoxymyoglobin. , 1998, Biochemistry.

[53]  J. Andersen,et al.  Crystal structures of a nitric oxide transport protein from a blood-sucking insect , 1998, Nature Structural Biology.

[54]  J. Andersen,et al.  Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus. , 1997, Biochemistry.

[55]  M. Hoshino,et al.  Studies on the Reaction Mechanism for Reductive Nitrosylation of Ferrihemoproteins in Buffer Solutions , 1996 .

[56]  J. S. Hyde,et al.  Permeability of nitric oxide through lipid bilayer membranes. , 1996, Free radical research.

[57]  S. Boxer,et al.  Trans effects in nitric oxide binding to myoglobin cavity mutant H93G. , 1996, Biochemistry.

[58]  R. Larsen,et al.  Ligand photolysis and recombination of Fe(II)protoporphyrin IX complexes in tetramethylene sulfoxide , 1995 .

[59]  J. A. Guimarães,et al.  Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus. , 1995, Biochemical Journal.

[60]  R. Pilz,et al.  Basis of guanylate cyclase activation by carbon monoxide. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[61]  V. Palaniappan,et al.  Acid-induced transformations of myoglobin. Characterization of a new equilibrium heme-pocket intermediate. , 1994, Biochemistry.

[62]  F. Walker,et al.  Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. , 1993, Science.

[63]  J. Doucet,et al.  Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex. , 1992, Biochemistry.

[64]  T. Traylor,et al.  Why nitric oxide , 1992 .

[65]  D. Wink,et al.  Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects. , 1991, Journal of medicinal chemistry.

[66]  D. Morikis,et al.  Spectroscopic studies of myoglobin at low pH: heme structure and ligation. , 1991, Biochemistry.

[67]  L. Ignarro.  Biosynthesis and metabolism of endothelium-derived nitric oxide. , 1990, Annual review of pharmacology and toxicology.

[68]  S. Strauss,et al.  Modeling low-pH hemoproteins. , 1989, The Journal of biological chemistry.

[69]  Thomas G. Spiro,et al.  Biological applications of Raman spectroscopy , 1987 .

[70]  T. Spiro,et al.  Alternative carbon monoxide binding modes for horseradish peroxidase studied by resonance Raman spectroscopy. , 1986, Biochemistry.

[71]  A. English,et al.  Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states. , 1986, Biochemistry.

[72]  Y. Nishimura,et al.  The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam. , 1985, The Journal of biological chemistry.

[73]  P. Stein,et al.  Porphyrin core expansion and doming in heme proteins. New evidence from resonance Raman spectra of six-coordinate high-spin iron(III) hemes , 1979 .

[74]  Y. Kyōgoku,et al.  Nature of the iron-ligand bond in ferrous low spin hemoproteins studied by resonance Raman scattering. , 1976, Journal of the American Chemical Society.

[75]  P. George,et al.  The oxidation of myoglobin to metmyoglobin by oxygen. III. Kinetic studies in the presence of carbon monoxide, and at different hydrogen-ion concentrations with considerations regarding the stability of oxymyoglobin. , 1954, The Biochemical journal.