Adduct formation between sulfite and the flavin of phototrophic bacterial flavocytochromes c. Kinetics of sequential bleach, recolor, and rebleach of flavin as a function of pH

The kinetics of sulfite adduct formation with the bound flavin in flavocytochromes c from the purple phototrophic bacterium Chromatium vinosum and the green phototrophic bacterium Chlorobium thiosulfatophilum have been investigated as a function of pH. Both species of flavocytochrome c rapidly react with sulfite to form a flavin sulfite adduct (k = 10(3)-10(5) M-1 s-1) which is bleached at 450-475 nm and has associated charge-transfer absorbance at 660 nm. The rate constant for adduct formation in flavocytochrome c is 2-4 orders of magnitude faster than for model flavins of comparable redox potential and is likely to be due to a basic residue near the N-1 position of the flavin, which not only raises the redox potential but also stabilizes the negatively charged adduct. There is a pK for adduct formation at 6.5, which suggests that the order of magnitude larger rate constant at pH 5 as compared to pH 10 in flavocytochrome c is due the influence of another positive charge, possibly a protonated histidine residue. The adduct is indefinitely stable at pH 5 but decomposes (the flavin recolors) in a first-order process accelerating above pH 6 (at pH 10, k = 0.1 s-1). The pK for recoloring is 8.5, which is suggestive of a cysteine sulfhydryl. On the basis of the observed pK and available chemical information, we believe that recoloring is due to a secondary effect of the reaction of sulfite with a protein cystine disulfide, which is adjacent to the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)