Quantifying redox dynamics of c-type cytochromes in a living cell suspension of dissimilatory metal-reducing bacteria

Abstract

To quickly and accurately quantify the redox dynamics of c-type cytochromes (c-Cyts) in a living cell suspension, diffusetransmission UV/visible (DT-UV/Vis) and normal UV/Vis spectroscopy were used to record spectra of c-Cyts in living Shewanella oneidensis MR-1 bacteria. DT-UV/Vis showed a higher absorbance of c-Cyts and lower background compared with normal UV/Vis, because interference from cell surface scattering was removed. The extinction coefficients of oxidized c-Cyts (410 nm) and reduced c-Cyts (419 and 552 nm) were observed. Using this method and the obtained c-Cyts extinction coefficients, the redox transformation kinetics of c-Cyts under anoxic conditions were successfully examined in the presence of various electron acceptors, including 9,10-anthraquinone-2,6-disulfonic acid, Cr(VI), Fe(III) citrate and oxygen. Therefore, the in situ spectral analysis of outer-membrane proteins of intact cells using DT-UV/Vis spectroscopy appears a promising method for investigating microbial metal reduction processes in living cell systems.