Mass Spectrometry of the Subunits of Highly Active Cytochrome b6f Complex from Spinach and the Cyanobacterium, M. laminosus: Identification of PetA,B,C,D,G,L,M,N Subunits in both Complexes and FNR in the Spinach b6f Complex

Abstract

Highly active cytochrome b6f complexes from spinach and the cyanobacterium M. laminosus have been analyzed by liquid-chromatography with electrospray-ionization mass spectrometry (LC-MS). Both size-exclusion and reverse-phase separations were used to separate proteins from other contaminants allowing measurement of protein molecular weights to an accuracy exceeding 0.01 % (+/- 3Da at 30000 Da). Four small subunits corresponding to the products of petG, petL, petM and petN were detected in both complexes. The M. laminosus complex had four larger subunits corresponding to the products of petA, petB, petC and petD, a total of eight subunits. The spinach complex had the same four large subunits and a fifth polypeptide of molecular weight 35314.0 Da, a total of nine subunits. Based upon the mass of CNBr fragments found in a fraction collected simultaneously to the elution of this component we identified the fifth polypeptide as ferredoxin-NADP oxidoreductase (FNR). This is the largest number and range of integral membrane subunits determined by MS techniques in an integral membrane protein. The excellent resolution afforded by ESI-MS revealed profiles that define the native covalent state of the components of the complex as well as their molecular heterogeneity. Supported by NIH AI-12601-24 (JW), NIH GM-18457 (WAC), and the WM Keck Foundation.