Proton homonuclear correlated spectroscopy as an assignment tool for hyperfine-shifted resonances in medium-sized paramagnetic proteins: cyanide-ligated yeast cytochrome c peroxidase as an example.
Two types of homonuclear proton COSY experiments are shown to be useful in making resonance assignments in cyanide-ligated cytochrome c peroxidase, a 34 kDa paramagnetic heme protein. Both magnitude COSY and phase-sensitive COSY experiments provide spectra useful for making proton assignments to resonances of strongly relaxed hyperfine-shifted protons. This initial investigation demonstrates that COSY experiments combined with NOESY experiments are feasible for hyperfine-shifted protons of paramagnetic proteins larger than metmyoglobins and ferricytochromes c, for which the nuclear spin-lattice relaxation times are in the range 70-300 ms. Taken together, COSY and NOESY experiments, although not yet widely applied to paramagnetic metalloproteins, provide a reliable protocol for accurately assigning hyperfine-shifted resonances that are part of a metalloenzyme's active site. Specific examples of expected proton homonuclear COSY connectivities that were not observed in these experiments are presented, and utilization of COSY with respect to the proton resonance line widths and apparent nuclear relaxation times is discussed. The COSY experiments presented here provide valuable verification of previously proposed hyperfine resonance assignments for cyanide-ligated cytochrome c peroxidase, which were made by using NOESY experiments alone, and in several instances expand these assignments to additional protons in particular amino acid spin systems.
Satterlee
J D
JD
Department of Chemistry, Washington State University, Pullman 99164-4630.
Russell
D J
DJ
Erman
J E
JE
D003486:Cyanides / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013329:Structure-Activity Relationship
D003486:Cyanides; D006418:Heme; D003578:Cytochrome-c Peroxidase