Why is the first assassin in the city not available for missions

Keep in mind that you'll only be attacked if your Templar Awareness meter hits max and stays there for a given amount of time. Just kill officials and bribe heralds every time you do something to raise it, and you'll never have to worry about being attacked in the first place.

(One thing to note that's a big difference from previous games - renovating property seems to add about 25% to your meter! It's really easy to max out if you're not careful. Also, liberating a den in the first place seems to max it out every time, but you seem to have a few minutes to lower it before you risk an attack.)

No. Not for regular missions or even if you station them in a city. They will remain den masters there and your dens will be protected. Though keep in mind, if they are killed in their missions, the den will (probably) be unprotected and will have to assign another one to the respective den and train to master. I don't know what happens in this case but you probably won't have to do the associated missions again.