I am about to submit my first app to the iTunes App Store. The docs say that the application needs to be submitted with a "512x512 px, 72ppi, RGB, flattened, no transparency JPEG image".
I have a couple of questions:
RGB means to ensure that you save the image as an RGB (rather than CMYK) JPEG. This is very likely to be the case already. (You can change the colour mode via the 'Image -> Mode' menu in Photoshop if you have the icon in the CMYK colour space for some reason. That said, all images destined for on screen usage should remain with the RGB colour space.)
As you say, directly saving as a JPEG (or doing save for web) will flatten the layers for you.
Whatever colo(u)r you want it to be. (i.e: It'll appear as saved.)
Hope that makes sense.