Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R

Unambiguous determination of metal atom oxidation state in an intact metall oprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic a bundance mass distributions for one or more holoprotein charge states. The iron atom oxidation state is determined unequivocally as Fe(III) for each o f four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobi n, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution- phase oxidation state is conserved following electrospray to produce gas-ph ase ions). However, the same Fe(III) oxidation state in all four heme prote ins is observed after prior reduction by sodium dithionite to produce Fe(II ) heme proteins in solution: thus proving that oxygen was present during th e electrospray process. Those results bear directly on the issue of similar ity (or lack thereof) of solution-phase and gas-phase protein conformations . Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holopro teins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe( II)heme from the covalently bound heme in cytochrome c. (C) 2000 American S ociety for Mass Spectrometry.