multifrequency epr spectroscopy of solar energy harvesting ......metals in biology: applications of...
TRANSCRIPT
MultifrequencyEPR Spectroscopy of
Solar Energy harvesting Species
ACERT 2009Friday, February 26, 2010
Part 1: Multifrequency ESEEM of Metal
ClusterNitrogen Ligands
• the “exact cancellation condition” drives a need for multifrequency pulsed EPR
• applications to mitoNEET Fe-S, Mn Catalase, PSII OEC
Friday, February 26, 2010
Exact cancellation Limit
• 1970s Mims and Peisach, 1980s David Singel and coworkers
• For nuclei with significant isotropic hyperfine coupling (A), best results when the external field matches the hyperfine field
• for a given g-value, this field matching determines the desired microwave frequency...instrumentation has lagged theory
Friday, February 26, 2010
Exact Cancellation LimitS=1/2, I=1 Case N14
ElectronZeeman
NuclearZeeman Hyperfine
m = -1/2s
m = +1/2s
NuclearQuadrupole
Friday, February 26, 2010
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
Three frequencies ν0, ν−, and ν+ givequadrupolar parameters e2qQ and η
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
Three frequencies ν0, ν−, and ν+ givequadrupolar parameters e2qQ and η
Then νdq provides a good estimate of thehyperfine coupling A
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.
(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Friday, February 26, 2010
UC DavisCalEPR Center
Bruker ECS 106 X-Band (9 GHz) CW
X/Ku-Bands (8-18 GHz) Pulsed
Ka-Band (31 GHz) Pulsed
D-Band (130 GHz) Pulsed
Bruker E580 X/Q-Bands (9, 34 GHz) CW/Pulsed
Friday, February 26, 2010
lab-built X/Ku-band (8-18 GHz, 1kW pulsed)
Friday, February 26, 2010
Ka-band 31 GHz (100W pulse/cw power)
Friday, February 26, 2010
Bruker E580 X (9 GHz)and Q (34 GHz) bands
Friday, February 26, 2010
MitoNeet FeS Protein
Mark L. Paddock, Patricia Jennings, UCSD
Rachel Nechushtai, Hebrew University
Michelle Dicus, Stefan Stoll, UC Davis
FeS protein that interacts witha family of insulin sensitizing drugs
(thiazolidinedione (TZD))
Friday, February 26, 2010
MitoNEET: Mitochondrial Outer MembraneProtein with a dimer of 2Fe2S Centers
with 3Cys-1His Ligation
Friday, February 26, 2010
“Original” X-band Rieske ESEEM (spinach b6f complex)
Britt et al., (1991)Biochem. 30:1862-1901(from my grad school homebuiltwith Melvin Klein)
Reiske 2Fe-2S has two histidinenitrogen ligands, more strongly coupled than exact cancellation limit at X-band
Friday, February 26, 2010
Ka Band ESEEM of MitoNEET
Friday, February 26, 2010
N15
Ka Band ESEEM of MitoNEET
Friday, February 26, 2010
N15
Ka Band ESEEM of MitoNEET
Friday, February 26, 2010
N15
Ka Band ESEEM of MitoNEET
N14
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Principal Investigator/Program Director(Last, First, Middle): Britt, R. David
Research Planν−
ν0
ν+
νsq
νdq
A Specific Aims
The transition metal Manganese plays a variety of im-portant roles in biology and medicine. Of particular in-terest is the unique water oxidation chemistry enabledby the tetranuclear Mn cluster of the oxygen evolvingcomplex of photosystem II. This cluster couples the highoxidation potential of a proximal tyrosine radical (Y •
Z ) tothe oxidation of two bound waters, releasing molecu-lar oxygen at the final step of a 5-intermediate cycle.Thus this system is an important example of metallo-radical chemistry, and the fact that each state can begenerated in high yield with laser flashes makes thisphotosynthetic system ideal for exploring this intrigu-ing chemistry. We will examine the intermediates ofthe oxygen evolving cycle with multifrequency pulsedEPR/ENDOR, using instruments at 9, 31, 35, and 130GHz frequencies.
B Progress Report
B.1 Publications during Review Period
Strickler, M. A., L. M. Walker, W. Hillier, R. D. Britt,and R. J. Debus. (2007) No Evidence from FTIRDifference Spectroscopy That Aspartate-342 of theD1 Polypeptide Ligates a Mn Ion That UndergoesOxidation during the S0 to S1, S1 to S2, or S2 toS3 Transitions in Photosystem II. Biochemistry 46,3151-3160.
Stich, T. A., S. Lahiri, G. Yeagle, M. Dicus, M. Brynda,A. Gunn, C. Aznar, V. J. DeRose, and R. D. Britt.(2007) Multifrequency Pulsed EPR Studies of Bio-logically Relevant Manganese(II) Complexes. Ap-plied Magnetic Resonance, 31, 321-341.
Yeagle, G. J., M. L. Gilchrist, Jr., L. Walker, R. J. De-bus, and R. D. Britt. (2008) Multifrequency Elec-tron Spin Echo Envelope Modulation Studies of Ni-trogen Ligation to the Manganese Cluster of Pho-tosystem II. Phil. Trans. Roc. Soc. B-BiologicalSciences 363, 1157-1166.
Strickler, M. A., H. J. Hwang, R. L. Burnap, J. Yano,L. M. Walker, R. J. Service, R. D. Britt, W. Hillier,and R. J. Debus. (2008) Glutamate-354 of CP43polypeptide interactions with the oxygen-evolvingMn4Ca cluster of photosystem II: a preliminarycharacterization of the Glu354Gln mutant. Phil.Trans. Soc. B-Biological Sciences 363, 1179-1187.
Yeagle, G. J., R. McCarrick, M. L. Gilchrist, and R. D.Britt. (2008) A Multifrequency Pulsed EPR Studyof the S2 State of the Photosystem II Mn Cluster.Inorganic Chemistry 47, 1803-1814.
Derbyshire, E., A. Gunn, M. Ibrahim, T. Spiro, R.Britt, and M. Marletta. (2008) Characterization ofTwo Different 5-Coordinate Soluble Guanylate Cy-clase Ferrous Nitrosyl Complexes. Biochemistry47, 3892-3899.
Brynda, M., R. D. Britt, Manganese in photosynthe-sis. In Biological Magnetic Resonance, v29 (2008)Metals in Biology: Applications of High ResolutionEPR to Metalloenzymes.
B.2 EPR Instrumentation
During this past year we have put a great deal of effortinto mastering the capabilities of our new Bruker E580pulsed EPR spectrometer with its X -band (9 GHz) andQ-band (34 GHz) capabilities. There have been somemechanical failures with the Q-band probe, which hasbeen sent back to Germany for repair twice, and it stilldisplays a large background EPR signal at low temper-ature that complicates our studies of the Mn signals ofthe OEC. Still, we work with it the best we can.
Last month we finally took delivery of the long over-due field-sweepable magnet for our D-band (130 GHz)instrument. This cryogen free, cryocooler-based su-perconducting magnet is required to perform variablefield studies of the PSII Mn cluster and other interestingparamagnetic metal systems. Figure 1 shows the newmagnet with the D-band spectrometer reinstalled.
PHS 398/2590 (Rev. 09/04) Page 1 Continuation Format Page
Friday, February 26, 2010
CW X Band EPR
Friday, February 26, 2010
Ka Band EPR and ESEEM (inset)
Friday, February 26, 2010
Ka Band ESEEMN15N14
Friday, February 26, 2010
Ka Band ESEEM
Q Band ESEEM
N15
N15
Friday, February 26, 2010
ESEEM
Q Band HYSCORE
N15
N15
Friday, February 26, 2010
53
14N 15N Aiso (MHz) –6.25(7) +8.77(10) T (MHz) –0.94(7) +1.32(10) Ax (MHz) Ay (MHz) Az (MHz) ! (°) " (°) # (°)
–5.28(7) –5.35(7) –8.13(7) +105(180) +57(2) +54(2)
+7.4(1) +7.5(1) +11.4(1) +105(180) +57(2) +54(2)
e2Qq/h (MHz) –2.47(4) $ +0.38(4) Px (MHz) Py (MHz) Pz (MHz) ! (°) " (°) # (°)
+0.38(2) +0.85(2) –1.24(2) –22(2) +51(2) +66(2)
Table 1. Experimentally determined 14N and 15N hyperfine and nuclear quadrupolar
coupling parameters of the N! nitrogen of His87 in human mitoNEET. Euler angles
describe the tensor orientations relative to the frame of the g-tensor.
Q Band ENDORN15
Friday, February 26, 2010
53
14N 15N Aiso (MHz) –6.25(7) +8.77(10) T (MHz) –0.94(7) +1.32(10) Ax (MHz) Ay (MHz) Az (MHz) ! (°) " (°) # (°)
–5.28(7) –5.35(7) –8.13(7) +105(180) +57(2) +54(2)
+7.4(1) +7.5(1) +11.4(1) +105(180) +57(2) +54(2)
e2Qq/h (MHz) –2.47(4) $ +0.38(4) Px (MHz) Py (MHz) Pz (MHz) ! (°) " (°) # (°)
+0.38(2) +0.85(2) –1.24(2) –22(2) +51(2) +66(2)
Table 1. Experimentally determined 14N and 15N hyperfine and nuclear quadrupolar
coupling parameters of the N! nitrogen of His87 in human mitoNEET. Euler angles
describe the tensor orientations relative to the frame of the g-tensor.
ESEEM N14
Friday, February 26, 2010
Current tensor orientation model (JACS submitted)[want to extend to single crystal studies]
Friday, February 26, 2010
Mn Catalase
Jim Penner-Hahn, U of Michigan
Jim Whittaker, OHSU
Troy Stich, UC Davis
Friday, February 26, 2010
!"#$%&'()* +,)(-"(+./0001+./021(34#%.5%.'6'(!%-%7%6'
Stemmler, T.L., et al. J. Am. Chem. Soc. 899:, 119, 9215.
X;<%.=(>($?76'(@)@@+
!=A
!"
!#
!$
Dinuclear Mn catalaseStudying Mn(III)Mn(IV) form
-relevance to PSII Mn
!"#$%&'()* +,)(-"(+./0001+./021(34#%.5%.'6'(!%-%7%6'
Stemmler, T.L., et al. J. Am. Chem. Soc. 899:, 119, 9215.
X;<%.=(>($?76'(@)@@+
!=A
!"
!#
!$
Friday, February 26, 2010
Mn1 Mn2
K162
E155
H188E70H73
E36
Mn1 Mn2R147
E148
H181E66H69
E35
Figure 1.
LP MnCat TT MnCat
Now have crystal structures from two organismsBoth show one histidine ligand to each Mn
Lactobacillus plantarum Thermus thermophilus
Friday, February 26, 2010
MHzFrequency [MHz]1086420
µs
1.151.101.05Field [T]
T + ! [µs]
N1
N2
exp
Frequency [MHz]
N1
N2
exp
* *9.77 GHz 30.67 GHz
400350300Magnetic Field [mT]
121086420
sim
N1
N2
exp
sim
LP MnCat
T + ! [µs]
N1
N2
exp
sim
543210
20151050
sim
Figure 2.
Lactobacillus plantarum
-10 -5 0 5 100
2
4
6
8
10
12
!1 [MHz]
-10 -5 0 5 100
2
4
6
8
10
12
!1 [MHz]
! 2[MHz]
! 2[MHz]
(N2 !dq",N2 !dq# + !0)(N2 !dq",N2 !dq# + !$)(N2 !dq",N2 !dq# + !+)
(N2 !dq",N2 !dq#)
(!0, !dq)(!$, !dq)(!+, !dq)
Figure 3.
X and Ka Band ESEEM and sims
X Band HYSCORE and sims
Aiso = −5.75 MHzAaniso = [−0.2 −0.6 +0.8] MHze2qQ = 2.01 MHz, η = 0.79 Euler angles α = 75° β = 0° γ = −10°
Aiso = +2.67 MHzAaniso = [+0.41 +0.15 −0.57] MHze2qQ = 2.25 MHz, η = 0.58 Euler angles α = 110°, β = 15°, γ = 85°
N1
N2
Friday, February 26, 2010
T + ! [µs]
N1
N2
exp
sim
121086420
Frequency [MHz]
T + ! [µs]
N1
N2
exp
Frequency [MHz]
N1
N2
exp
N1
N2
exp
* *30.67 GHz
1.151.101.05Field [T]
9.77 GHz
Figure 4
400350300Magnetic Field [mT]
543210
sim
TT MnCat
1086420 20151050
sim
Figure 5.
-10 -5 0 5 100
2
4
6
8
10
12
-10 -5 0 5 100
2
4
6
8
10
12
!1 [MHz]!1 [MHz]
! 2[MHz]
! 2[MHz]
(!", !dq)(!+, !dq)
(N2 !dq#,N2 !dq$ + !+)
(N2 !dq#,N2 !dq$)
Thermus thermophilus
X and Ka Band ESEEM and sims
X Band HYSCORE and sims
N1
N2
Aiso = +2.28 MHzAaniso = [+0.42 +0.28 −0.70] MHz,e2qQ = 2.29 MHz, η = 0.50 Euler angles α = 0° β = 50° γ = 50°
Aiso = −5.2 MHzAaniso = [−0.3 −0.7 +1.0] MHze2qQ = 2.0 MHz, η = 0.4Euler angles α = 70° β = 60° γ = 80°
Friday, February 26, 2010
Photosystem IIOEC Manganese
Richard Debus, UC Riverside
Greg Yeagle, Troy Stich, UC Davis
Friday, February 26, 2010
D1-His332
D1-His337
Ca
MnD
MnC MnA
MnB
B
D1-Asp170
D1-His332
D1-His337
D1-Glu333
D1-Tyr161
CP43-Glu354
D1-Asp342MnA
D1-Glu189
D1-Asp170
MnD
A
D1-Ala344
Ca
MnB
MnC
Summary of current X-ray picture of ligands
Superposition ofdifferent structures
Friday, February 26, 2010
3-pulse multifrequency ESEEM of the Photosystem II S2-state Mn “multiline” signal in the X/Ku band instrument
specific labelling (with Bruce Diner)showed these to be histidine N transitions15
Friday, February 26, 2010
76543210T + ! [!S]
20151050Frequency [MHz]
sim
exp
sim
exp
sim
exp
simexp
Ka-band
Q-band
Ka-band
Q-band
14N-PSII from Synechocystis
sim
exp
Ka-band
Q-band
Ka-band
Q-band
76543210T + ! [!S]
sim
exp
20151050Frequency (MHz)
sim
exp
sim
exp
15N-PSII from Synechocystis
Ka and Q band ESEEM and Simulations
N and N15 14
Friday, February 26, 2010
76543210T + ! [!S]
20151050Frequency [MHz]
sim
exp
sim
exp
sim
exp
simexp
Ka-band
Q-band
Ka-band
Q-band
14N-PSII from Synechocystis
sim
exp
Ka-band
Q-band
Ka-band
Q-band
76543210T + ! [!S]
sim
exp
20151050Frequency (MHz)
sim
exp
sim
exp
15N-PSII from Synechocystis
14 Aiso(14N) = 6.89 MHzAaniso(14N) = [1.14 0.56 -1.70] MHz e2Qq/h = -1.98 MHz, eta = 0.84
N simulation parameters
Friday, February 26, 2010
D1-His332
D1-His337
Ca
MnD
MnC MnA
MnB
B
D1-Asp170
D1-His332
D1-His337
D1-Glu333
D1-Tyr161
CP43-Glu354
D1-Asp342MnA
D1-Glu189
D1-Asp170
MnD
A
D1-Ala344
Ca
MnB
MnC
Nearly identical spectra fromspinach PSII ( N)
(Yeagle et al, Inorg Chem 2008)
X-Ku results simulated with same parameters, no evidence for
multiple nitrogens
Which histidine is it?
14
Friday, February 26, 2010
Part 2: HF-EPR and Substrate/Cofactor Radical Electronic
Structure
• Single crystal study of biliverdin radical intermediate
• pterin radical of nitric oxide synthase
Friday, February 26, 2010
Biliverdin Radicals in Light Harvesting/
Sensing Pigment Synthesis
Stefan Stoll, Alex Gunn, Clark Lagarias, Andy Fisher, UC Davis
Andrew Ozarowski, NHMFL
Friday, February 26, 2010
C-phycocyanin
disk formed as a trimer of alpha/beta heterodimers
2.5nm
Friday, February 26, 2010
Phycocyanobilin:Ferredoxin Oxidoreductase
(pcyA)
Biliverdin
Phycocyanobilin
RadicalIntermediates
in Vinyl Reduction
Friday, February 26, 2010
D-band 130 GHz(100mW pulse power)
“Cryogen-free” 0-8 Tesla Magnet
Friday, February 26, 2010
Friday, February 26, 2010
PcyA: high-field EPR of crystals and powdersStoll et al, JACS 2009 131 1986
Friday, February 26, 2010
PcyA: DFT predictions of g tensor
Friday, February 26, 2010
Pterin Radicals in NO Synthesis
Stefan Stoll, Yaser NejatyJahromy UC Davis
Josh Woodward, Michael Marletta UC Berkley
Andrew Ozarowski, NHMFL
Friday, February 26, 2010
These structures inform the discussion of the mechanismsof NOS in a number of interesting ways. The hydrogen-bonding network seems to orient arginine and NHA rigidlywith respect to the heme as shown in Figure 3. It is imme-diately apparent that NHA is bound as the antistereoisomer, a fact that has not been previously discussed.This is noteworthy because the hydroxylimine oxygen andthe guanidinium carbon of the NHA are both distant fromthe heme iron (4.3 Å and 4.4 Å, respectively). Thisarrangement presents significant difficulties for some ofthe proposed mechanisms of NOS discussed below.
Characteristics of the NOS reactionsThe NOS reaction generates citrulline by oxidizing L-Argthrough the intermediacy of NHA. However, the identity ofthe nitrogen oxide product, NO, or the nitroxyl anion, NO–,was only determined recently. Several reports suggested thatthe nitroxyl anion, NO–, was generated by NOS and con-verted to NO under the catalysis of superoxide dismutase(SOD) [28,29]. Recently, however, NO from the NOS reac-tion was directly detected in the absence of SOD [30]. Therehas been a debate regarding the stoichiometry of NADPHand the amount of citrulline generated in the NOS reaction.The controversy arises because several NADPH-consumingreactions occur along with the NOS reaction. These sidereactions include the generation of superoxide ion (O2
–) bythe NOS heme [31•,32] and the oxidation of NADPH byperoxynitrite generated from NO and O2
– [28]. Hevel andMarletta [9] have correctly determined the NADPH/cit-rulline stoichiometry by conducting the reaction withH4B-saturated iNOS in the presence of SOD. Anotherimportant characteristic of the NOS reaction products is theoxygen source for NO and the urea-oxygen of citrulline.Using 16O-labeled NHA under 18O2 conditions, the ureaoxygen of citrulline was found to contain exclusively 18O,
whereas the NO product originated completely from the N-hydroxy group of NHA [33,34].
The N-hydroxylation of L-Arg consumes one equivalenteach of NADPH and O2, and is typical of a P450 oxygenasereaction. The generally accepted P450 reaction mechanismcan account for the stoichiometry and the product forma-tion [35]. Marletta et al. [36] have proposed anon-P450-type mechanism to account for the oxidation ofL-Arg in which O2 activation by an H4B and a non-hememetal ion was proposed to furnish the intermediate oxidiz-ing L-Arg to generate NHA. This mechanism is analogousto that of the pterin-dependent aromatic amino acid hydro-genases [37]. However, this notion has been challenged bytwo recent discoveries. First, the 5-methyl H4B analogsupports the NOS reaction, but it does not support O2 acti-vation [38]. Secondly, catalytic activity analysis of amutated NOS, in which the histidine residues near theheme group were substituted, and metal ion analysis bothsuggest that there is no catalytic role for a non-heme redox-active metal in NOS [39].
In the second step of the NOS reaction, the heme group acti-vates O2 by recruiting only one reducing equivalent fromNADPH and, apparently, one from NHA. A key questionthat needs to be answered is the order of the redox eventsinvolved in the oxidation of NHA. Particularly, it is unclearwhether the NOS Fe(III)-heme is reduced by NHA or the
Nitric oxide synthase: models and mechanisms Groves and Wang 689
Table 1
Redox potentials of NOS cofactors, NHA derivatives andvarious iron porphyrin oxidation states.
Redox couples Redox potentials* Reference
OxoFe(IV)(P+•)/oxoFe(IV)P !1.4~1.6 V [67]
OxoFe(IV)P/Fe(OH)P !1.0~1.3 V [67]
NHA/NHA-derived iminoxy radical !710 mV (pH 7.5) [8]
H4B/qH2B 150~180 mV [68]
SuperoxoFe(III)/peroxoFe(III)heme 10 mV [69]
NHA–/NHA•† !–200 mV [57]
NOS Fe(III)/Fe(II)heme –248~–263 mV [70]
FAD/FAD+• –270~–290 mV [71]
NADPH –324 mV [72]
*E1/2 versus NHE, at pH 7 aqueous solution except where otherwisecited. †Estimated from the redox potential of the amidoximate/amidoxyliminoxy radical couple. NHA–, oximate derived from the N-hydroxygroup of NHA; NHA•, the iminoxy radical derived from NHA;qH2B, quinonoid dihydrobiopterin.
Figure 3
Schematic structure of the active site of NHA-bound murine iNOS.NHA is shown in bold.
NN
NNFe
OHO
O
O
S
N
N
NH
O
N OH
H2N
H
HO
Cys194
N
N
N
O2CH
H2N
ON
NO
O
N O
R
OH
H
H
H
O
N
N
O
III
Trp366
Glu371
H
Gly365
4.0Å
R!
HOH
H
OH
H
H
H
H
H
Current Opinion in Chemical Biology
Friday, February 26, 2010
X band CW EPR
Friday, February 26, 2010
X band CW EPR
Friday, February 26, 2010
High Field CW EPR
Friday, February 26, 2010
Q Band Davies ENDOR
Friday, February 26, 2010
g = [2.0043 2.00355 2.00215]
A_H6 = [42 45 51]A_N5 = [0.1 0.1 65]A_N8 = [0.1 0.1 15]A_H5 = [-9 -45 -30]A_H8 = [-3 -15 -10]
EPR Parameters Constrained by Simultaneous Fit to All Spectra
Friday, February 26, 2010
DFT Calculations SupportsCation Radical Assignment:H6 resonance for example
Friday, February 26, 2010
Stefan StollStefan Stoll
Michelle Dicus Greg YeagleKa band, mitoNEET Ka band, PSII
Catalase, PSII mitoNEET, RadicalsDr. Troy Stich Dr. Stefan Stoll
Yaser NejatyJahromyNOS
Alex GunnpcyA
Friday, February 26, 2010
The End
Friday, February 26, 2010