The suf operon helps bacteria survive under adverse conditions used by human body to fight against bacterial invasion, such as iron starvation and oxidative stress. Hence knowledge of the biochemical mechanism of the suf pathway may help design new antibacterial drugs against certain pathogenic bacteria. The role of two key proteins from the suf operon, SufA and SufD, is studied here. SufA and SufD were purified using column chromatography. UV-Vis spectroscopy and measurements of iron and sulfide were used to confirm a 2Fe-2S cluster present on SufA as purified from in vivo expression. Point mutations and growth studies were performed to characterize the role of SufD. The SufD_H128A point mutation abolished the function of the entire Suf pathway as shown by in vivo growth experiments in a suf operon deletion strain. The SufD_H128A mutation caused accumulation in vivo of the 2Fe-2S cluster on SufA, resulting in increased levels of holo-SufA during purification. Whole-cell Mössbauer spectroscopy confirmed increased 2Fe-2S cluster signal from intact bacterial cells containing the SufD_H128A point mutant relative to control cells. Our results show that SufA contains a 2Fe-2S cluster in vivo and that point mutations in the SufD protein can alter the level of Fe-S cluster in SufA. These findings represent an important step towards understanding the role of SufA and SufD in Fe-S cluster biogenesis.