Abstract
Recent reports indicate that oxidized cobalamin, Cbl(III), can interfere with the biological effects of nitric oxide (NO) on vascular and visceral smooth muscle and in other systems. In attempting to elucidate the mechanism of these effects of Cbl(III), we reported that a Cbl(III)NO complex could be detected by electron paramagnetic resonance (EPR) spectroscopy, but not by ultraviolet/visible spectroscopy. Subsequently, others concluded that the alleged Cbl(III)NO complex is detectable by ultraviolet/visible, but not by EPR spectroscopy and provided ultraviolet/visible evidence for an alleged Cbl(II)NO complex. We report further investigation of the interaction of NO with Cbl, using both techniques, Fourier transform infrared (FTIR) spectroscopy and mass spectrometry. Our EPR results and the UV/VIS results of others appear to be experimental artifacts that can now, at least in part, be explained. Under conditions where FTIR measurements readily detect a N-O stretching frequency of NO bound to Fe(II), we do not detect a similar signal that can be ascribed to either Cbl(III)NO or Cbl(II)NO, indicating that neither Cbl(III) nor Cbl(II) form a stable complex with NO. Loss of the Cbl(II) EPR signal and mass spectral detection of N2O upon addition of NO to Cbl(II) solutions, demonstrates that Cbl(II), which is present in aerobic Cbl(III) solutions, reduces NO; however, this reaction does not appear to be fast enough to account for the observed biological effects in aerated media. Nitric oxide also reacts rapidly and irreversibly with the superoxo complex of Cbl(III), Cbl(III)O2−, which is always present in aerated solutions of Cbl(III). We believe that this latter reaction accounts for the observed inactivation of NO by Cbl(III) in biological systems. Because Cbl(III)O2− is spontaneously regenerated from Cbl(II) and O2 in aerated solutions, this may constitute a cyclic mechanism for the rapid elimination (oxidation) of NO. Thus, several physicochemical techniques fail to provide convincing evidence for the existence of stable Cbl(III)NO or Cbl(II)NO complexes but do provide evidence that Cbl species participate in redox reactions with NO under aerobic conditions, thereby inhibiting its physiological roles.
Footnotes
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Send reprint requests to: Dr. Roger P. Smith, Department of Pharmacology and Toxicology, Dartmouth Medical School, 7650 Remsen, Room 519, Hanover, NH 03755-3835.
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↵1 This work was supported in part by Grant HL 14127 from the National Heart, Lung and Blood Institute (R.P.S.). The Bruker ESP 300 spectrometer was purchased with funding from the NSF (Grant CHE-8701406).
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↵2 Current address: Cystic Fibrosis Pulmonary Research, 7013 Thurston Bowls, University of North Carolina, Chapel Hill, NC 27599.
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↵3 Current address: Mount Hood Community College, Science Division, 26000 SE Stark, Gresham, OR 97030.
- Abbreviations:
- Cbl(III)
- oxidized cobalamin
- Cbl(II)
- reduced cobalamin
- AdoCbl
- adenosylcobalamin
- MeCbl
- methylcobalamin
- NO
- nitric oxide
- Hb
- deoxyhemoglobin
- Mb
- myoglobin
- MetHb
- methemoglobin
- EPR
- electron paramagnetic resonance
- FTIR
- Fourier transform infrared
- GSH
- reduced glutathione
- GSSG
- oxidized glutathione
- UV/VIS
- ultraviolet/visible
- NO2−
- nitrite
- O2−
- superoxide
- N2O
- nitrous oxide
- Received June 2, 1997.
- Accepted January 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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