You are assuming that they still lose to Pitt after that loss. Pretty big assumption, IMO.
That's true, because you really do make different decisions when you're a one-loss team (if the playoff is your goal) than if you're highly ranked and have a slight margin for error. They might have gone for it on some fourth downs or other stuff in that situation.
Of course, everyone here is arguing based on ALABAMA assumptions - the national championship. But there might possibly be another one that had more of a long-term effect than any other: the final play of the LSU-Auburn game is going to affect the sport and the SEC for years.
If LSU wins that game then Auburn is 1-3, and I honestly think that Malzahn is on the hottest of seats although I doubt they fire him at that point in time. When LSU fired Miles the next day, it set off a series of dominoes that led to Ed Orgeron winding up a head coach in the SEC (where he already failed once), got Cam Cameron fired (putting him in line for possibly being the Tide OC), led to season-long rumors about Lane Kiffin going POSSIBLY to LSU as the new OC, and enabled Leonard Fournette to sit out a bowl game. Rumors also erupted about Tom Herman to LSU, which set off a bidding war with UT and LSU for an unproven at that level head coach.
People forget that LSU was only three plays from being in the national title discussion. One could also argue that their pick at the end of the Wisconsin game had a snowball effect.
Another big play was the fourth down failed conversion by Louisville vs Clemson (as already noted). It would have been VERY interesting to see whether Ohio State or Clemson got picked in that circumstance......or both......and then Penn State is probably in the tournament as well.
"Single biggest play" is like "greatest player," the context of what you're asking matters. If you're talking about "what play contributed the most to the eventual national championship winner" then we can take our pick (pardon the pun) from several plays in the Alabama-Clemson game. If you mean "most significant overall," you have to define "overall" and that can be something other than "who won it all."
The last play of the LSU-Auburn game not only involved those teams, it got other teams dragged into "what if" scenarios regarding coaches, both head and coordinators. It got Auburn's kicker some national press, saved Malzahn for now, blew Miles apart, got Orgeron a trial job, got Cam Cameron fired, set in motion Fournette sitting out the Citrus Bowl, got Herman, Jimbo Fisher, Lane Kiffin awash in coaching rumors, and helped contribute to the demise of Charlie Strong at Texas (I do think he was probably gone anyway but the fact that LSU might steal Herman from them undoubtedly made that call all that much easier).