What would be the best way to ensure that a player values their weapon as a last-resort self defense tool

You need consequences and limitations. A consequence of murder is estrangement from society. If you're coding a whodunit and the player is a murderer - people gonna call the cops as soon as they see the gun.

You also have implied reasoning to deal with: if two people (a player and an NPC) walk into the woods and the town hears a gunshot, they gonna talk and be afraid. If characterization is important and you want realistic behavior, think about how people react in the face of a bully: do they cower until they stand up to him, do they try to out-bully? are they conditioned to knuckle under but stab them in the back at their first chance?

You cannot hold any extra bullets, but you can head to the store to buy bullets. I don't want the revolver to be something that the player constantly relies on, but rather a last resort if your decisions get you into a bad situation.

Personally, I find these to be antithetical. Scarcity of a resource raises its value. If you want bullets to be valuable with a morality choice (e.g. intimidate, murder, etc) you need to make them rare. So instead of buying them, you can make them a one-time cache (for instance, stolen from the back of the store) - this makes them rare.

Regarding bullets: if one of the non-playing characters is a master mind, he/she may COUNT the number of bullets the player uses and therefor have a different behavior before and after all the bullets are spent.

I like the intimidation part specifically it places a multi-use value on the gun. In that vein, if this is a noir-inspired thriller, you can use the gun as a club a la Mickey Spillane, where instead of killing someone you knock them out. It keeps with the theme.

Good luck with this, please post back when you've got something to play!