The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.