If it's an easy question, the lifelines order is irrelevant - the public will get it right anyway.
If it's a hard question, by using Ask the Audience first, as you said, will spread out the "guessers" and the right answer should get more chances to get a higher vote %, than if there were 2 choices only. Even if there are trickster choices that make it look like it's the right answer, which leads most guessers to choose it, these tend to stay after the 50-50, so it'd be still better to go for Ask the Audience first, where at least some of the guessers would still be spread out by the other 2 wrong choices.
One advantage in choosing 50-50 first would be that some people could be undecided between one wrong choice and the right choice, and with the 50-50, the wrong one could be eliminated and they'd guess the right choice. Others could also be sure that one of the choices wasn't certainly right, and if that one stayed after the 50-50, they would then also guess the right one. But again, the 50-50 almost always keeps the 2 strongest choices, so I'd still go for Ask the Audience first and 50-50 afterward.
Edit: I am changing my conjecture, you should pick 50-50 first then the audience. you will get guessers+people that know vs just guessers. the right option will always get more votes.
Using Ask the Audience first would still be better, the amount of guessers on a hard question will be too high to get any statistically significant result with only 2 choices.