Nitric oxide synthase (NOS) catalyzes the production of nitric oxide from L-arginine and dioxygen at a thiolate-ligated heme active site. Although many of the reaction intermediates are as yet unidentified, it is well established that the catalytic cycle begins with substrate binding and rate-limiting electron transfer to the heme. Here we show that Ru(II)-diimine and Re(I)-diimine electron tunneling wires trigger nanosecond photoreduction of the active-site heme in the enzyme. Very rapid generation of a reduced thiolate-ligated heme opens the way for direct observation of short-lived intermediates in the NOS reaction cycle.