You should be using the top PCIE slot for the GPU. I know logically one could see the second slot as x16 and think it's better, but it's not. The second slot is coming from the PLX switch which actually only has an x8 link to the CPU on this particular motherboard. The first slot has a native x8 link to the CPU when other slots are populated. Using the second slot would just add latency, though rather negligibly so. The way I would would do it is have the GPU in the first slot, and the other two cards (looks like audio and Wifi) in the black slots. That would completely avoid the PLX PEX8747 switch. Your GPU would still only be x8 but it could cut down on latency to the GPU and the card in the black x8 slot.