Corynebacterium diphtheriae utilizes heme and hemoglobin (Hb) as iron sources for growth in low-iron environments. In C. diphtheriae， the two-component signal transduction systems (TCSs) ChrSA and HrrSA are responsive to Hb levels and regulate the transcription of promoters for hmuO， hrtAB， and hemA ChrSA and HrrSA activate transcription at the hmuO promoter and repress transcription at hemA in an Hb-dependent manner. In this study， we show that HrrSA is the predominant repressor at hemA and that its activity results in transcriptional repression in the presence and absence of Hb， whereas repression of hemA by ChrSA is primarily responsive to Hb. DNA binding studies showed that both ChrA and HrrA bind to the hemA promoter region at virtually identical sequences. ChrA binding was enhanced by phosphorylation， while binding to DNA by HrrA was independent of its phosphorylation state. ChrA and HrrA are phosphorylated in vitro by the sensor kinase ChrS， whereas no kinase activity was observed with HrrS in vitro Phosphorylated ChrA was not observed in vivo， even in the presence of Hb， which is likely due to the instability of the phosphate moiety on ChrA. However， phosphorylation of HrrA was observed in vivo regardless of the presence of the Hb inducer， and genetic analysis indicates that ChrS is responsible for most of the phosphorylation of HrrA in vivo Phosphorylation studies strongly suggest that HrrS functions primarily as a phosphatase and has only minimal kinase activity. These findings collectively show a complex mechanism of regulation at the hemA promoter， where both two-component systems act in concert to optimize expression of heme biosynthetic enzymes.